Detection of Cystic Fibrosis Transmembrane Conductance ......Karan Malhotra Master of Science...
Transcript of Detection of Cystic Fibrosis Transmembrane Conductance ......Karan Malhotra Master of Science...
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic
Acid Hybridization Assay and a Smartphone Camera
by
Karan Malhotra
A thesis submitted in conformity with the requirements for the degree of Master of Science
Department of Chemistry University of Toronto
copy Copyright by Karan Malhotra 2018
ii
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic
Acid Hybridization Assay and a Smartphone Camera
Karan Malhotra
Master of Science
Department of Chemistry
University of Toronto
2018
Abstract
Diagnostic technology that utilizes paper substrates and device cameras offers opportunities for
development of cost effective point-of-care technologies The translation of assays operating in
aqueous solution require further development for implementation in paper substrates This report
presents and compares two methods for determination of oligonucleotides that serve as indicators
of Cystic Fibrosis differentiating wild type and mutant type sequences containing a 3-base
deletion The transduction strategy operates by selective hybridization of dye-labelled
oligonucleotides (target or reporters) to capture probes immobilized on quantum dots and
hybridization results in emission of dyes via resonance energy transfer Detection is based on
hybridization of fluorophore labelled target or hybridization of unlabelled target and labelled
reporter in a sandwich assay format Selectivity to determine mismatched sequences required
control of stringency conditions using formamide as a chaotrope It was determined that both
formats can distinguish between wild type and mutant type samples on paper substrates
iii
Acknowledgments
I would like to begin by expressing my gratitude to Professor Ulrich J Krull for his guidance and
mentorship throughout my graduate career I am privileged to have worked in his lab with other
motivated and driven graduate students I have learned a lot at my time in the Chemical Sensors
Group (CSG) and I will always cherish my experience and memories here I am also grateful to
Professor Aaron R Wheeler who has graciously agreed to be the second reader for this thesis I
would also like to acknowledge Professor Paul A E Piunno with whom I have had many
conversations about research graduate work and judo Special thanks are also extended to Dr M
Omair Noor for his mentorship I have learned a lot about research from him that I would have
never have learned otherwise
I am grateful to numerous members of the University of Toronto Community for their help
Members of the UTM stores Microelectronics Academic workshop are sincerely thanked for their
support I would also like to thank the administrative staff at the UTM campus for their support
during my graduate work including Carmen Bryson Jessica Bailey Michelle Bae Christina M
Fortes and Roxana Moreira-Diaz
Much of the work in this thesis would not have been possible without the support of members from
the Department of Chemical and Physical Science and CSG I am forever grateful to Dr Abootaleb
Sedighi Dr Samer Doughan Dr Peter Mitrakos Dr Sreekumair Nair Dr Thottackad
Radhakrishnan Anna Shahmuradyan Yi Han Phillip Rolo Hifza Najib Muhammad Shahrukh
David Hrovat Richard Fuku Hamna Fayyaz and Alex Escobar for their support
Lastly I would like to acknowledge my family and friends for their continued support I would
like to express my gratitude to my sister brother-in-law and baby niece for always making life
enjoyable I would also like to thank my girlfriend for her continued support throughout my time
in grad school Finally none of this would have been possible without the sacrifice and
encouragement from my parents I am truly blessed to have you as my role models
iv
Table of Contents
Acknowledgments iii
Table of Contents iv
List of Tables vi
List of Figures vii
Chapter 1 1
Introduction 1
11 Cystic Fibrosis and Genes Associated with Cystic Fibrosis Transmembrane Protein 1
111 Nucleic Acids based Detection of Genes Associated with CF and ∆F508
mutation of CFTR Gene1
12 Nucleic Acids and Oligonucleotide Detection 3
121 Structure and Composition of DNA Hybridization 4
122 Thermodynamics of DNA Hybridization 5
123 Notes and Considerations for POC Application 7
13 Quantum dots 8
131 Quantum Confinement and The Particle in a Box 10
14 Fluorescence and Resonance Energy Transfer 11
141 Fluorescence Resonance Energy Transfer (FRET)11
15 Paper Based Analytical Devices 14
151 Paper Substrates for Sensing Technology Overview 15
152 Cellulose Modification and Smartphone-based Detection 15
16 Thesis Objectives and Contributions 17
Chapter 2 19
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation
Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera 19
21 Experimental 19
211 Methods20
v
212 Instrumentation 24
22 Results and Discussion 25
221 FRET Pair Characterization (gQD ndash Cy3) 25
222 Oligonucleotide Hybridization in Solution 26
223 Oligonucleotide Hybridization in Paper Substrates 28
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by
Smartphone Imaging 32
225 Analytical Figures of Merit 38
226 Selectivity for Mixtures of WT and MT Targets 40
227 Paper-based Assay Response for Complex Sample Matrices 42
228 Blind Assay for Detection and Quantification of CFTR Target Mixes 43
Chapter 3 45
Conclusion and Future Work 45
31 Future Directions 46
References 47
vi
List of Tables
Table 1 Summary of FDA approved Nucleic Acid Based Tests for Diagnosis of CF 2
Table 2 Oligonucleotide Sequences used in Hybridization Assays 20
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids 34
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids 34
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids 34
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids 34
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids 36
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids 36
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids 36
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids 37
Table 11 Analytical Performance Direct and Sandwich Bioassays 40
Table 12 Blind Assay for Direct and Sandwich Assays 44
vii
List of Figures
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and Crick
Arrows on the ribbons represent the directionality bias for the single strands and dimensions for
the polymer are presented with one turn of the helix every 34 nm the distance between base pairs
every 034 nm and the distance between the phosphate backbone and the central axis every 1 nm
B shows the hydrogen bonding taking place between complementary pairs of nucleobases as
proposed by Chargaff with adenine (A) having two hydrogen bonds with thymine (T) and guanine
(G) having three hydrogen bonds with cytosine (C) Image was adapted with permission
Copyright Nature Education 201331 5
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of ZnS
B Quantum dots of different colors are presented with their corresponding core size image of
solution and photoluminescence spectra and color C Diagram representing the quantum
confinement and the change in band gap energy as material size decreases below the Bohr-exciton
radius Here CB and VB represent the conduction and valence band respectively and Eg represent
the band gap energies Image adapted with permission Copyright 2011 American Chemical
Society60 9
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of colloidally
stable and spherical QD (green) with multiple FRET acceptors (yellow) (b) Change in FRET
efficiency based on changes in the distance between donor and acceptor (c) QD (green)
immobilized on a surface can interact with multiple FRET acceptors by interacting with adjacent
acceptors Image acquired with permission from Algar et al70 Copyright Elsevier 2010 12
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in blue)
are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3 on
the proximal end and upon hybridization is brought to proximity with gQDs allowing for FRET
to take place (B) In sandwich assay format the probe strand hybridizes with the target strand (seen
in red) such that there is an overhang on the distal end Reporter strand (seen in green) hybridizes
with the overhang region of the target strand bringing to proximity the Cy3 label on the proximal
end of the reporter 14
viii
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society of
Chemistry 2016 16
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A) Reaction
zones consisted of chemically modified paper that were conjugated with gQD-oligonucleotide
probes Zones contained WT and MT controls and test zones where unknown samples were
spotted and imaged Detection was based on the principle of RET with gQDs used as donors and
Cy3 labels on oligonucleotide strands as acceptors (B) Imaging used a smartphone camera with
data processing by ImageJ to split the image to RGB color channels 18
Figure 7 Image of buffer solution leakage from hydrophilic paper zones 23
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines and
emission is shown as solid lines 26
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by fluorescence
spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii) CFTR single
DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT target strands The
concentration-response curves for the different gQD-probe conjugates are shown A WT Cy3
labelled target strands are seen in blue and MT Cy3 labelled target strands are seen in orange
Normalized PL spectra for the calibration curves are shown for B) CFTR WT Cy3 labelled target
strands and C) CFTR MT Cy3 labelled target strands ( indicates increasing target concentration)
27
Figure 10 Representations of the two different direct assay formats investigated in solution phase
gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA MT probe
and were mixed with complementary CFTR WT Cy3 target strands and CFTR MT Cy3 target
strands Hybridization resulted in proximity of gQDs and Cy3 which resulted in FRET 28
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of Cy3
labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol (vii) 75
ix
pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of spots that
may not be visible otherwise 29
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers to
WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash MT target
(D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and Tm) were calculated
using the nearest neighbor method3839 30
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash MT target
(D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and Tm) were calculated
using the nearest neighbor method3839 32
Figure 14 Determination of optimal wash conditions for direct and sandwich assay considered
RG Ratios with variation of formamide concentration for wash times of 0 5 10 15 and 20 min
The optimal wash conditions for direct assay was found to be BB+10F for 5 minutes for (A)
gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal wash conditions for
sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-WT probe sequence and
BB+5F for 20 minutes for (D) gQD-MT probe sequence 38
Figure 15 Concentration-response curves showing the RG ratiometric response of the direct and
sandwich assay formats (Ai) gQD-WT probe conjugates were used for determination of Cy3
labelled WT targets and (Bi) gQD-MT probe conjugates were used for determination of Cy3
labelled MT targets (Ci) gQD-WT probe conjugates were used for determination of unlabelled
WT targets with Cy3 labelled reporters and (Di) gQD-MT probe conjugates were used for
determination of unlabelled MT targets with Cy3 labelled reporters The RG ratiometric response
of the direct assay at the low pmol concentration range was also determined (Aii) gQD-WT probe
conjugates and (Bii) gQD-MT probe conjugates The sandwich assay format (Cii) gQD-WT probe
conjugates and (Dii) gQD-MT probe conjugates Note that the scale for (A) and (B) is logarithmic
Each error bar represents one standard deviation for n=4 replicates 39
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes and
(Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined using
background corrected RG ratio plots for hybridization of gQD-probe conjugates with Cy3 labelled
x
targets (for direct assay A and B) and gQD-probe conjugates with unlabeled targets and Cy3
labelled reporter sequences (for sandwich assay C and D) Response of the hybridization assay
was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-wash (Bi and Di) MT
probe conjugates Post-wash assays yielded signal response shown in Aii and Cii for WT probe
conjugates and in Bii and Dii for MT probe conjugates Error bars represent one standard deviation
for n = 4 replicates 41
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates and
(B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to direct assay
and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was collected for (C)
gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars represent one standard
deviation for n = 4 replicates 42
1
Chapter 1
Introduction
11 Cystic Fibrosis and Genes Associated with Cystic Fibrosis Transmembrane Protein
Cystic fibrosis (CF) is a multi-system fatal autosomal recessive disorder that is
characterized by viscous secretions in the lungs of patients due to mutations in cystic fibrosis
transmembrane conductance regulator protein (CFTR) CF affects 1 in 3000 births with ~70000
people affected worldwide1ndash5 Over 1500 mutations for the CFTR protein have been found but few
are common and fewer result in the disease Of the few mutations responsible for the disease state
the deletion of phenylalanine at the 508 position (∆F508) is responsible for over two-thirds of the
cases while all other mutations account for no more than 5 of the cases individually256
Development of sensing technology for early detection of ∆F508 would serve to enable improved
screening by clinicians to identify the predominant gene carriers The strategies for diagnosing CF
are based on newborn screening (NBS) programs that work via screening for serum markers
including the immunoreactive trypsinogen (IRT) assay7ndash9 This assay is typically followed by
diagnosis of the genetic basis of disease including detection of ∆F508 and related mutations based
on determining the presence of specific oligonucleotide sequences Finally a sweat chloride test
is performed to diagnose patients with CF All of these techniques require skilled technicians to
process samples perform and analyse tests via resource-intensive technologies10 The aim of this
work is to contribute to the development of a low cost easy to use and portable method for sensing
CFTR ∆F508 gene mutations beginning with a focus on a suitable transduction strategy
111 Nucleic Acids based Detection of Genes Associated with CF and ∆F508 mutation of CFTR Gene
There are multiple strategies for transducing the presence of genes associated with CF and
some of the technologies that have been approved by the United Stated Food and Drug
Administration (FDA) for use as in-vitro medical devices are presented in Table 1 (accessed Feb
20th 2018)11
2
Table 1 Summary of FDA approved Nucleic Acid Based Tests for Diagnosis of CF
Manufacturer Trade Name Detection Strategy
Illumina Inc Illumina MiSeqDx Cystic
Fibrosis Clinical Sequencing
Assay
Next-gen sequencing by
synthesis
Illumina MiSeqDx Cystic
Fibrosis 139-Variant Assay
Luminex Molecular
Diagnostics Inc
xTAG Cystic Fibrosis 60 kit v2 Microbead-dye barcode
coupled microarray analysis xTAG Cystic Fibrosis 39 kit v2
Osmetech Molecular
Diagnostics
eSensor CF Genotyping Test Sandwich hybridization assay
with ferrocene tag for cyclic
voltammetry analysis
Nanosphere Inc Verigene CFTR and Verigene
CFTR PolyT Nucleic Acid Tests
Genomic amplification
followed by sandwich assay
with probes and gold
nanoparticle reporters for
analysis
Third Wave Technology Inc InPlex CF Molecular Test PCR coupled with FRET based
microwell plate
Celera Diagnostics Cystic Fibrosis Genotyping
Assay
PCR coupled with capillary
electrophoresis and
oligonucleotide ligation assay
Typically these technologies require the use of specialized facilities and dedicated
technicians for analysis of patient samples and confirmation of CF may take up to a few weeks79
The resources and time required to diagnose patients may be reduced through the development of
point-of-care (POC) devices In particular the use of paper-based test strips with smartphone
detection for on-site rapid screening of disease markers would serve to alleviate the burden placed
on the health care system by more expensive techniques12
At the core of POC technology is the transduction strategy and much effort has gone into
developing optical13 and electrochemical methods14 for generating and measuring signal Yet the
application of this technology has not been investigated for selective sensing of similar nucleic
acid sequences that are often found to be associated with the genetic basis of disease Thus to
further discuss the challenges in this field it is important to address some of the background
technology that has been developed for POC sensors In particular this chapter will discuss nucleic
acid detection and the thermodynamics associated with hybridization interactions the use of
3
formamide as a chaotrope for controlling hybridization the use of nanomaterials like quantum dots
as integrated components in the bioassays for fluorescence resonance energy transfer-based
sensing strategies and the application of paper as a platform and substrate for sensing
12 Nucleic Acids and Oligonucleotide Detection
Deoxyribonucleic acid (DNA) is a class of biopolymers that stores hereditary information
and ribonucleic acid (RNA) functions as the set of instructions for synthesis of proteins15 The two-
step process by which the DNA nucleobase sequence is transcribed for production of RNA and
subsequently RNA is used as a template for translation to produce proteins is referred to as the
central dogma of molecular biology16 Proteins carry out the function that is encoded in the genetic
regions of DNA by interacting with other molecules and biopolymers present within and on the
surface of cells The specific interactions that govern the proteinsrsquo function are due to the three-
dimensional structure of the amino acid sequence that composes proteins17 The order of amino
acids which composes proteins is based on the nucleobase sequence of transcribing RNA (and
therefore DNA) Thus hereditary information stored as the base sequence of DNA can govern the
sequence of amino acids and therefore the structure and function of proteins1617 There are
numerous types of diseases that have arisen due to nucleobase-pair mutations in the sequence of
gene coding regions of DNA18 Mutations of DNA bases influence the amino acid sequence that
compose proteins and a three-base pair deletion like the one found for phenylalanine at codon 508
significantly alters the function of the protein associated with the CFTR gene Other types of
genetic diseases also arise due to mutations of the base pair sequence associated with DNA and
strategies for detection of nucleic acid mutations offer a method to detect the presence of a disease
state
To determine the genetic basis of disease for guiding clinical treatment diagnostic
technology for sensing nucleic acids must be further developed The main goal of clinical
diagnostic technology is to determine the molecular basis of disease for guiding patient therapy
because knowledge obtained from diagnostics are paramount for programing treatment strategies
Clinical diagnostics using a POC strategy offers improved opportunity for wide-scale screening
due to the advantages of low cost ease of manufacturing ease of transport ease of use and ease
of disposal19 One approach to the detection of genetic materials (deoxyribonucleic acid) is based
4
on hybridization and this process requires consideration of the chemical composition structure
and thermodynamics associated with hybridization
121 Structure and Composition of DNA Hybridization
Elucidation of DNArsquos structure and function has a long-storied history that has impacted
many fields of research including chemistry biology and medicine Much of the early work
related to DNA was focused on the structure of DNA with scientists focusing on the key details
related to the chemical composition of the monomers and the structural format of the polymeric
structure of DNA20ndash24 The key findings of the structure of DNA are summarized as follows
1 The structure for the DNA salt is composed of two helical polymer chains that are
coiled around one another and around a shared axis (see Figure 1A) The outside of the
chains is composed of phosphate-sugars groups and the chains are linked together on
the inside via hydrogen bonds between the nucleotide bases
2 The chains are anti-parallel in conformation with ie the 5rsquo end of one chain is bound
via the nucleobases to the 3rsquo end of the other chain
3 Both chains follow a right-handed helix (one type of DNA known as Z-DNA follows
a left-handed helix but this was discovered later)25 and base residues are present on the
chains every 34 Å with structural repeats every ten residues The distance from the
central shared axis to the phosphorous atom is 10 Å
4 The four bases composing DNA bond to a specific pair (see Figure 1B) ie adenine
(purine) binds with thymine (pyrimidine) and guanine (purine) binds with cytosine
(pyrimidine) The relationship of molar equivalency between pairs of bases ie A ndash T
and G ndash C was determined earlier by Chargaff in 195026
Details related to the structure and composition of DNA has formed the basis of our
understanding of the role of DNA in molecular and cell biology Through the structure of DNA
the mechanism for DNA replication27 transcription28 and translation29 for protein synthesis was
elucidated The confirmation of DNA as the storage for hereditary information paved the way for
initiatives like the Human Genome Project and insights from this undertaking have fueled research
regarding the genetic basis of disease30
5
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and
Crick Arrows on the ribbons represent the directionality bias for the single strands and
dimensions for the polymer are presented with one turn of the helix every 34 nm the
distance between base pairs every 034 nm and the distance between the phosphate
backbone and the central axis every 1 nm B shows the hydrogen bonding taking place
between complementary pairs of nucleobases as proposed by Chargaff with adenine (A)
having two hydrogen bonds with thymine (T) and guanine (G) having three hydrogen bonds
with cytosine (C) Image was adapted with permission Copyright Nature Education 201331
122 Thermodynamics of DNA Hybridization
Design and development of DNA-based technologies have been guided by the
thermodynamic modelling of hybridization Techniques like PCR3233 and isothermal
amplification34 rely on accurate control over the annealing of primers and DNA sensors often uses
temperature and chaotropic agents for achieving selectivity35 One of the useful tools for modelling
hybridization and mismatch-based interactions is the nearest neighbor method (NN)36 To explore
the strategy between the NN method and hybridization of DNA it is useful to understand some
details related to predicting the melting temperature (Tm)
First the system of interest will be defined at the equilibrium of dsDNA and ssDNA at the
point where both populations are equal ie half the strands of DNA are in the double helix state
and the other half are single-stranded and are often in various conformations Tm is the temperature
6
at which this equilibrium is found Next the equilibrium constant Keq is defined as being a ratio
of the concentration of dsDNA and ssDNA (as stated in Equation 1) A second expression can be
derived from the Vanrsquot Hoff equation (as stated in Equation 2) where ∆Ho and ∆So are the standard
enthalpy and entropy of hybridization and lnCT is the natural logarithm of the total strand
concentration This second equation can be used to calculate the thermodynamic parameters
related to Tm with the same being true vice versa37
Equation 1 = [][]
Equation 2 = ∆∆
With this foundation investigation into the NN method for modelling can be undertaken
The thermodynamics associated with a base pair are related to some degree with neighboring base
pairs Free energy values and other related parameters have been determined experimentally for
model oligonucleotide sequences This information is then used in conjunction with the nearest
neighbor algorithm (as presented in Equation 3) for obtaining the Tm for the strand of interest
Here base pair doublets are considered for sequence stability with ten unique combinations of
doublets (5rsquo-3rsquo) CG GC AT AA (also = TT) AG (also = CT) AC (also = GT) GA (also = TC)
GG (also = CC) TG (also = CA)38
Equation 3 ∆ = ∆ + ∆ + sum ∆
Equation 4 ∆ = ∆ minus ∆
In Equation 3 the ∆Gi(total) refers to the free energy of the strand of interest ∆G(init)
refers to the free energy of the strand of initiating base pair ∆G(sym) refers to the free energy of
symmetry Gj refers to the free energy associated with one of ten nearest neighbor stacking
interactions and nij is the appearance rate of the stacking interaction of interest Thermodynamic
parameters are also present for entropy and enthalpy allowing for the calculation of the Tm using
Equation 43638 Higher Tm values indicate greater stability than lower Tm values39ndash42 The NN
method can also be used along with a database of mismatch energetics to determine the
thermodynamic parameters related to those sequences
7
Tm values when used in conjunction with the free energies provide a theoretical basis for
designing probe ndash capture strand interactions This understanding can be useful when designing
wash conditions that control stringency for oligonucleotides composed of sequences with high
similarity Stringency control can be achieved using higher temperature (because increasing
temperature results in de-annealing of sequences and has greater effect on hybrids with partial
complementarity)43 by controlling the ionic strength of hybridization44 and via chaotropic agents
such as formamide45 and urea46 Although all strategies are valid for reducing false positive signals
(that arise from partially complementary strands of oligonucleotides) the use of washes containing
chaotropic agents may be more applicable for the POC given that temperature control requires a
temperature module
Chaotropic agents like formamide lower the melting temperature of duplex DNA by
engaging with the hydrogen bond network of DNA The degree by which temperature is lowered
depends on the GC content the conformations of single and duplex forms and the hydration state
of the strand (but typically can range 24 ndash 29 degC per mole of formamide)45 Chemically
formamide strongly associates with DNA is capable of four hydrogen bonds (same as water) and
is a stronger hydrogen bond acceptor than water Formamide ndash water bonds have been reported to
be 20 stronger than water ndash water bonds and it is accepted that formamide engages the hydration
network around DNA4547 Thus using formamide in washes for DNA hybridization can lower the
melting temperature favoring fully complementary hybrids over partially complementary hybrids
123 Notes and Considerations for POC Application
Developing a DNA screening device for the POC application requires consideration of the
many challenges faced by clinicians When screening genetic samples from blood it is important
to note that samples are often complex with proteins and other type of biomolecules (in addition
to cellular debris) and these materials may occlude the signal generated from target detection48
Another challenge to note for nucleic acid-based diagnostics is the low amount of target present in
clinical samples For example one milliliter of human blood contains approximately 107
leukocytes corresponding to femtomolar quantities (fM or attomoles 10-18 moles) of target nucleic
acid Thus detection strategies requiring hybridization-based assay require enzymatic
amplification of the target materials or improved analytical figures of merit for application in
POC49 Presently there are many different strategies for enzymatic amplification of nucleic acids
8
including polymerase chain reaction32 loop-mediated isothermal amplification34 helicase-
dependent isothermal amplification50 and recombinase polymerase amplification51 Post
amplification targets are often detected using hybridization-based assays using Watson-Crick base
pairing for detection of targets of interest Typically capture probes of complementary sequence
to targets are immobilized on a surface and the presence of target forms hybrids that are transduced
via electrochemical5253 or optical strategies54 Nano-surfaces can be integrated into this detection
strategy with oligonucleotides immobilized on the surface of nanoparticles allowing for
transduction via near-field phenomenon
13 Quantum dots
Nanomaterials based on gold and semiconductor composites have had a significant impact
across many different research fields including the chemical physical and biological sciences
Interest in nanoparticles has been driven due to the unique fundamental properties of these
materials as they approach and occupy size regions between bulk material and isolated atoms
Luminescent semiconductor-based quantum dots (QDs) in particular have attracted attention due
to their unique electro-optical properties arising from small size scales (typically ranging from
2 ndash 10 nm and consisting of 102 ndash 104 atoms per crystal) The key factors of interest for these
particles are material composition and size with a combination of the two giving rise to control of
physical properties such as the spectral profile and photon band gap energies55ndash59
There are many strategies for preparing and tuning the electro-optical properties of QDs
but some of the most studied from a synthetic perspective are based on binary composites of
elements from groups II-VI (like CdSe CdS or CdTe) and III-V (like InAs)55ndash58 For binary
composites luminescent properties can be controlled by choice of materials (selecting specific
regions of the UV-vis spectrum) and by control of size whereby smaller crystals are blue-shifted
and larger crystals are red-shifted5759 QDs used in most biological investigations are constructed
in a coreshell manner where the core is composed on a composite of materials previously
mentioned and the shell is composed of an inert coating (ie ZnS see Figure 2A) The QD shell
protects the nanoparticle from environmental degradation forming a protective layer and provides
a larger potential energy barrier for confining the exciton The shell material also provides a
synthetic strategy for controlling the core size and the type of shell allows for designing a class of
ligands for functionalizing the nanoparticle5556
9
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of
ZnS B Quantum dots of different colors are presented with their corresponding core size
image of solution and photoluminescence spectra and color C Diagram representing the
quantum confinement and the change in band gap energy as material size decreases below
the Bohr-exciton radius Here CB and VB represent the conduction and valence band
respectively and Eg represent the band gap energies Image adapted with permission
Copyright 2011 American Chemical Society60
The resulting particles have been incorporated into biological systems using surface ligands
with chemistry that allows the crystals to be stable in aqueous and physiological buffers6162
Further functionalization of these ligands has also allowed for the integration of biomolecules like
nucleic acids63 and proteins64 and polymers like polyethylene glycol (PEG) allowing applications
that extend from biological imaging65 to diagnostic device development and commercial
technologies566667 Optically quantum dots (QD) have broad absorption wavelengths (from the
UV into the visible) narrow and symmetrical emission photoluminescence (PL) profiles (25 nm
of full width at half maximum) high quantum yields and photochemical stability59 These
spectral properties in addition to the large surface area of QDs make them favourable donors for
RET processes
10
131 Quantum Confinement and The Particle in a Box
A brief overview of the quantum mechanics related to QDs will be discussed before
detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids
As the semiconducting material that composes QDs transitions from the bulk scale to the nano-
scale the valence and conductance bands of the semiconductor material split into discrete
energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the
composite of materials however for nanomaterials the band gap can also be tuned by modulating
the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the
dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like
CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an
electron-hole pair When an electron is excited by a photon of greater energy than the band gap
(the probability increases at higher energies yielding broad absorption spectra) the separation of
the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used
to describe this phenomenon is called quantum confinement and the model that best describes it is
the particle in a box575960
In this model a particle is said to be confined in a symmetrical box (of diameter a) where
the center of the box is denoted as = 0 and the edges of the box are denoted as = (
( Here
the potential energy inside the box +( le le
(- is said to be zero and the potential energy outside
the box + le ( ge
(- is said to be infinite The resulting probability of finding a particle outside
the confines of the box is zero 0 = 0 + le ( ge
(-1 and the discrete energy
eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of
interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material
The surface of the material represents the impenetrable barrier (potential energy is infinity)
restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869
As size of the QDs decreases the energy required for excitation increases because the
exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral
properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a
RET based system because the surface area of QDs allows for loading of multiple biomolecules
Thus multiple pathways of RET can take place that can collectively improve energy transfer
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
ii
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic
Acid Hybridization Assay and a Smartphone Camera
Karan Malhotra
Master of Science
Department of Chemistry
University of Toronto
2018
Abstract
Diagnostic technology that utilizes paper substrates and device cameras offers opportunities for
development of cost effective point-of-care technologies The translation of assays operating in
aqueous solution require further development for implementation in paper substrates This report
presents and compares two methods for determination of oligonucleotides that serve as indicators
of Cystic Fibrosis differentiating wild type and mutant type sequences containing a 3-base
deletion The transduction strategy operates by selective hybridization of dye-labelled
oligonucleotides (target or reporters) to capture probes immobilized on quantum dots and
hybridization results in emission of dyes via resonance energy transfer Detection is based on
hybridization of fluorophore labelled target or hybridization of unlabelled target and labelled
reporter in a sandwich assay format Selectivity to determine mismatched sequences required
control of stringency conditions using formamide as a chaotrope It was determined that both
formats can distinguish between wild type and mutant type samples on paper substrates
iii
Acknowledgments
I would like to begin by expressing my gratitude to Professor Ulrich J Krull for his guidance and
mentorship throughout my graduate career I am privileged to have worked in his lab with other
motivated and driven graduate students I have learned a lot at my time in the Chemical Sensors
Group (CSG) and I will always cherish my experience and memories here I am also grateful to
Professor Aaron R Wheeler who has graciously agreed to be the second reader for this thesis I
would also like to acknowledge Professor Paul A E Piunno with whom I have had many
conversations about research graduate work and judo Special thanks are also extended to Dr M
Omair Noor for his mentorship I have learned a lot about research from him that I would have
never have learned otherwise
I am grateful to numerous members of the University of Toronto Community for their help
Members of the UTM stores Microelectronics Academic workshop are sincerely thanked for their
support I would also like to thank the administrative staff at the UTM campus for their support
during my graduate work including Carmen Bryson Jessica Bailey Michelle Bae Christina M
Fortes and Roxana Moreira-Diaz
Much of the work in this thesis would not have been possible without the support of members from
the Department of Chemical and Physical Science and CSG I am forever grateful to Dr Abootaleb
Sedighi Dr Samer Doughan Dr Peter Mitrakos Dr Sreekumair Nair Dr Thottackad
Radhakrishnan Anna Shahmuradyan Yi Han Phillip Rolo Hifza Najib Muhammad Shahrukh
David Hrovat Richard Fuku Hamna Fayyaz and Alex Escobar for their support
Lastly I would like to acknowledge my family and friends for their continued support I would
like to express my gratitude to my sister brother-in-law and baby niece for always making life
enjoyable I would also like to thank my girlfriend for her continued support throughout my time
in grad school Finally none of this would have been possible without the sacrifice and
encouragement from my parents I am truly blessed to have you as my role models
iv
Table of Contents
Acknowledgments iii
Table of Contents iv
List of Tables vi
List of Figures vii
Chapter 1 1
Introduction 1
11 Cystic Fibrosis and Genes Associated with Cystic Fibrosis Transmembrane Protein 1
111 Nucleic Acids based Detection of Genes Associated with CF and ∆F508
mutation of CFTR Gene1
12 Nucleic Acids and Oligonucleotide Detection 3
121 Structure and Composition of DNA Hybridization 4
122 Thermodynamics of DNA Hybridization 5
123 Notes and Considerations for POC Application 7
13 Quantum dots 8
131 Quantum Confinement and The Particle in a Box 10
14 Fluorescence and Resonance Energy Transfer 11
141 Fluorescence Resonance Energy Transfer (FRET)11
15 Paper Based Analytical Devices 14
151 Paper Substrates for Sensing Technology Overview 15
152 Cellulose Modification and Smartphone-based Detection 15
16 Thesis Objectives and Contributions 17
Chapter 2 19
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation
Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera 19
21 Experimental 19
211 Methods20
v
212 Instrumentation 24
22 Results and Discussion 25
221 FRET Pair Characterization (gQD ndash Cy3) 25
222 Oligonucleotide Hybridization in Solution 26
223 Oligonucleotide Hybridization in Paper Substrates 28
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by
Smartphone Imaging 32
225 Analytical Figures of Merit 38
226 Selectivity for Mixtures of WT and MT Targets 40
227 Paper-based Assay Response for Complex Sample Matrices 42
228 Blind Assay for Detection and Quantification of CFTR Target Mixes 43
Chapter 3 45
Conclusion and Future Work 45
31 Future Directions 46
References 47
vi
List of Tables
Table 1 Summary of FDA approved Nucleic Acid Based Tests for Diagnosis of CF 2
Table 2 Oligonucleotide Sequences used in Hybridization Assays 20
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids 34
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids 34
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids 34
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids 34
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids 36
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids 36
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids 36
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids 37
Table 11 Analytical Performance Direct and Sandwich Bioassays 40
Table 12 Blind Assay for Direct and Sandwich Assays 44
vii
List of Figures
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and Crick
Arrows on the ribbons represent the directionality bias for the single strands and dimensions for
the polymer are presented with one turn of the helix every 34 nm the distance between base pairs
every 034 nm and the distance between the phosphate backbone and the central axis every 1 nm
B shows the hydrogen bonding taking place between complementary pairs of nucleobases as
proposed by Chargaff with adenine (A) having two hydrogen bonds with thymine (T) and guanine
(G) having three hydrogen bonds with cytosine (C) Image was adapted with permission
Copyright Nature Education 201331 5
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of ZnS
B Quantum dots of different colors are presented with their corresponding core size image of
solution and photoluminescence spectra and color C Diagram representing the quantum
confinement and the change in band gap energy as material size decreases below the Bohr-exciton
radius Here CB and VB represent the conduction and valence band respectively and Eg represent
the band gap energies Image adapted with permission Copyright 2011 American Chemical
Society60 9
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of colloidally
stable and spherical QD (green) with multiple FRET acceptors (yellow) (b) Change in FRET
efficiency based on changes in the distance between donor and acceptor (c) QD (green)
immobilized on a surface can interact with multiple FRET acceptors by interacting with adjacent
acceptors Image acquired with permission from Algar et al70 Copyright Elsevier 2010 12
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in blue)
are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3 on
the proximal end and upon hybridization is brought to proximity with gQDs allowing for FRET
to take place (B) In sandwich assay format the probe strand hybridizes with the target strand (seen
in red) such that there is an overhang on the distal end Reporter strand (seen in green) hybridizes
with the overhang region of the target strand bringing to proximity the Cy3 label on the proximal
end of the reporter 14
viii
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society of
Chemistry 2016 16
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A) Reaction
zones consisted of chemically modified paper that were conjugated with gQD-oligonucleotide
probes Zones contained WT and MT controls and test zones where unknown samples were
spotted and imaged Detection was based on the principle of RET with gQDs used as donors and
Cy3 labels on oligonucleotide strands as acceptors (B) Imaging used a smartphone camera with
data processing by ImageJ to split the image to RGB color channels 18
Figure 7 Image of buffer solution leakage from hydrophilic paper zones 23
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines and
emission is shown as solid lines 26
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by fluorescence
spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii) CFTR single
DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT target strands The
concentration-response curves for the different gQD-probe conjugates are shown A WT Cy3
labelled target strands are seen in blue and MT Cy3 labelled target strands are seen in orange
Normalized PL spectra for the calibration curves are shown for B) CFTR WT Cy3 labelled target
strands and C) CFTR MT Cy3 labelled target strands ( indicates increasing target concentration)
27
Figure 10 Representations of the two different direct assay formats investigated in solution phase
gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA MT probe
and were mixed with complementary CFTR WT Cy3 target strands and CFTR MT Cy3 target
strands Hybridization resulted in proximity of gQDs and Cy3 which resulted in FRET 28
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of Cy3
labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol (vii) 75
ix
pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of spots that
may not be visible otherwise 29
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers to
WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash MT target
(D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and Tm) were calculated
using the nearest neighbor method3839 30
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash MT target
(D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and Tm) were calculated
using the nearest neighbor method3839 32
Figure 14 Determination of optimal wash conditions for direct and sandwich assay considered
RG Ratios with variation of formamide concentration for wash times of 0 5 10 15 and 20 min
The optimal wash conditions for direct assay was found to be BB+10F for 5 minutes for (A)
gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal wash conditions for
sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-WT probe sequence and
BB+5F for 20 minutes for (D) gQD-MT probe sequence 38
Figure 15 Concentration-response curves showing the RG ratiometric response of the direct and
sandwich assay formats (Ai) gQD-WT probe conjugates were used for determination of Cy3
labelled WT targets and (Bi) gQD-MT probe conjugates were used for determination of Cy3
labelled MT targets (Ci) gQD-WT probe conjugates were used for determination of unlabelled
WT targets with Cy3 labelled reporters and (Di) gQD-MT probe conjugates were used for
determination of unlabelled MT targets with Cy3 labelled reporters The RG ratiometric response
of the direct assay at the low pmol concentration range was also determined (Aii) gQD-WT probe
conjugates and (Bii) gQD-MT probe conjugates The sandwich assay format (Cii) gQD-WT probe
conjugates and (Dii) gQD-MT probe conjugates Note that the scale for (A) and (B) is logarithmic
Each error bar represents one standard deviation for n=4 replicates 39
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes and
(Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined using
background corrected RG ratio plots for hybridization of gQD-probe conjugates with Cy3 labelled
x
targets (for direct assay A and B) and gQD-probe conjugates with unlabeled targets and Cy3
labelled reporter sequences (for sandwich assay C and D) Response of the hybridization assay
was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-wash (Bi and Di) MT
probe conjugates Post-wash assays yielded signal response shown in Aii and Cii for WT probe
conjugates and in Bii and Dii for MT probe conjugates Error bars represent one standard deviation
for n = 4 replicates 41
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates and
(B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to direct assay
and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was collected for (C)
gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars represent one standard
deviation for n = 4 replicates 42
1
Chapter 1
Introduction
11 Cystic Fibrosis and Genes Associated with Cystic Fibrosis Transmembrane Protein
Cystic fibrosis (CF) is a multi-system fatal autosomal recessive disorder that is
characterized by viscous secretions in the lungs of patients due to mutations in cystic fibrosis
transmembrane conductance regulator protein (CFTR) CF affects 1 in 3000 births with ~70000
people affected worldwide1ndash5 Over 1500 mutations for the CFTR protein have been found but few
are common and fewer result in the disease Of the few mutations responsible for the disease state
the deletion of phenylalanine at the 508 position (∆F508) is responsible for over two-thirds of the
cases while all other mutations account for no more than 5 of the cases individually256
Development of sensing technology for early detection of ∆F508 would serve to enable improved
screening by clinicians to identify the predominant gene carriers The strategies for diagnosing CF
are based on newborn screening (NBS) programs that work via screening for serum markers
including the immunoreactive trypsinogen (IRT) assay7ndash9 This assay is typically followed by
diagnosis of the genetic basis of disease including detection of ∆F508 and related mutations based
on determining the presence of specific oligonucleotide sequences Finally a sweat chloride test
is performed to diagnose patients with CF All of these techniques require skilled technicians to
process samples perform and analyse tests via resource-intensive technologies10 The aim of this
work is to contribute to the development of a low cost easy to use and portable method for sensing
CFTR ∆F508 gene mutations beginning with a focus on a suitable transduction strategy
111 Nucleic Acids based Detection of Genes Associated with CF and ∆F508 mutation of CFTR Gene
There are multiple strategies for transducing the presence of genes associated with CF and
some of the technologies that have been approved by the United Stated Food and Drug
Administration (FDA) for use as in-vitro medical devices are presented in Table 1 (accessed Feb
20th 2018)11
2
Table 1 Summary of FDA approved Nucleic Acid Based Tests for Diagnosis of CF
Manufacturer Trade Name Detection Strategy
Illumina Inc Illumina MiSeqDx Cystic
Fibrosis Clinical Sequencing
Assay
Next-gen sequencing by
synthesis
Illumina MiSeqDx Cystic
Fibrosis 139-Variant Assay
Luminex Molecular
Diagnostics Inc
xTAG Cystic Fibrosis 60 kit v2 Microbead-dye barcode
coupled microarray analysis xTAG Cystic Fibrosis 39 kit v2
Osmetech Molecular
Diagnostics
eSensor CF Genotyping Test Sandwich hybridization assay
with ferrocene tag for cyclic
voltammetry analysis
Nanosphere Inc Verigene CFTR and Verigene
CFTR PolyT Nucleic Acid Tests
Genomic amplification
followed by sandwich assay
with probes and gold
nanoparticle reporters for
analysis
Third Wave Technology Inc InPlex CF Molecular Test PCR coupled with FRET based
microwell plate
Celera Diagnostics Cystic Fibrosis Genotyping
Assay
PCR coupled with capillary
electrophoresis and
oligonucleotide ligation assay
Typically these technologies require the use of specialized facilities and dedicated
technicians for analysis of patient samples and confirmation of CF may take up to a few weeks79
The resources and time required to diagnose patients may be reduced through the development of
point-of-care (POC) devices In particular the use of paper-based test strips with smartphone
detection for on-site rapid screening of disease markers would serve to alleviate the burden placed
on the health care system by more expensive techniques12
At the core of POC technology is the transduction strategy and much effort has gone into
developing optical13 and electrochemical methods14 for generating and measuring signal Yet the
application of this technology has not been investigated for selective sensing of similar nucleic
acid sequences that are often found to be associated with the genetic basis of disease Thus to
further discuss the challenges in this field it is important to address some of the background
technology that has been developed for POC sensors In particular this chapter will discuss nucleic
acid detection and the thermodynamics associated with hybridization interactions the use of
3
formamide as a chaotrope for controlling hybridization the use of nanomaterials like quantum dots
as integrated components in the bioassays for fluorescence resonance energy transfer-based
sensing strategies and the application of paper as a platform and substrate for sensing
12 Nucleic Acids and Oligonucleotide Detection
Deoxyribonucleic acid (DNA) is a class of biopolymers that stores hereditary information
and ribonucleic acid (RNA) functions as the set of instructions for synthesis of proteins15 The two-
step process by which the DNA nucleobase sequence is transcribed for production of RNA and
subsequently RNA is used as a template for translation to produce proteins is referred to as the
central dogma of molecular biology16 Proteins carry out the function that is encoded in the genetic
regions of DNA by interacting with other molecules and biopolymers present within and on the
surface of cells The specific interactions that govern the proteinsrsquo function are due to the three-
dimensional structure of the amino acid sequence that composes proteins17 The order of amino
acids which composes proteins is based on the nucleobase sequence of transcribing RNA (and
therefore DNA) Thus hereditary information stored as the base sequence of DNA can govern the
sequence of amino acids and therefore the structure and function of proteins1617 There are
numerous types of diseases that have arisen due to nucleobase-pair mutations in the sequence of
gene coding regions of DNA18 Mutations of DNA bases influence the amino acid sequence that
compose proteins and a three-base pair deletion like the one found for phenylalanine at codon 508
significantly alters the function of the protein associated with the CFTR gene Other types of
genetic diseases also arise due to mutations of the base pair sequence associated with DNA and
strategies for detection of nucleic acid mutations offer a method to detect the presence of a disease
state
To determine the genetic basis of disease for guiding clinical treatment diagnostic
technology for sensing nucleic acids must be further developed The main goal of clinical
diagnostic technology is to determine the molecular basis of disease for guiding patient therapy
because knowledge obtained from diagnostics are paramount for programing treatment strategies
Clinical diagnostics using a POC strategy offers improved opportunity for wide-scale screening
due to the advantages of low cost ease of manufacturing ease of transport ease of use and ease
of disposal19 One approach to the detection of genetic materials (deoxyribonucleic acid) is based
4
on hybridization and this process requires consideration of the chemical composition structure
and thermodynamics associated with hybridization
121 Structure and Composition of DNA Hybridization
Elucidation of DNArsquos structure and function has a long-storied history that has impacted
many fields of research including chemistry biology and medicine Much of the early work
related to DNA was focused on the structure of DNA with scientists focusing on the key details
related to the chemical composition of the monomers and the structural format of the polymeric
structure of DNA20ndash24 The key findings of the structure of DNA are summarized as follows
1 The structure for the DNA salt is composed of two helical polymer chains that are
coiled around one another and around a shared axis (see Figure 1A) The outside of the
chains is composed of phosphate-sugars groups and the chains are linked together on
the inside via hydrogen bonds between the nucleotide bases
2 The chains are anti-parallel in conformation with ie the 5rsquo end of one chain is bound
via the nucleobases to the 3rsquo end of the other chain
3 Both chains follow a right-handed helix (one type of DNA known as Z-DNA follows
a left-handed helix but this was discovered later)25 and base residues are present on the
chains every 34 Å with structural repeats every ten residues The distance from the
central shared axis to the phosphorous atom is 10 Å
4 The four bases composing DNA bond to a specific pair (see Figure 1B) ie adenine
(purine) binds with thymine (pyrimidine) and guanine (purine) binds with cytosine
(pyrimidine) The relationship of molar equivalency between pairs of bases ie A ndash T
and G ndash C was determined earlier by Chargaff in 195026
Details related to the structure and composition of DNA has formed the basis of our
understanding of the role of DNA in molecular and cell biology Through the structure of DNA
the mechanism for DNA replication27 transcription28 and translation29 for protein synthesis was
elucidated The confirmation of DNA as the storage for hereditary information paved the way for
initiatives like the Human Genome Project and insights from this undertaking have fueled research
regarding the genetic basis of disease30
5
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and
Crick Arrows on the ribbons represent the directionality bias for the single strands and
dimensions for the polymer are presented with one turn of the helix every 34 nm the
distance between base pairs every 034 nm and the distance between the phosphate
backbone and the central axis every 1 nm B shows the hydrogen bonding taking place
between complementary pairs of nucleobases as proposed by Chargaff with adenine (A)
having two hydrogen bonds with thymine (T) and guanine (G) having three hydrogen bonds
with cytosine (C) Image was adapted with permission Copyright Nature Education 201331
122 Thermodynamics of DNA Hybridization
Design and development of DNA-based technologies have been guided by the
thermodynamic modelling of hybridization Techniques like PCR3233 and isothermal
amplification34 rely on accurate control over the annealing of primers and DNA sensors often uses
temperature and chaotropic agents for achieving selectivity35 One of the useful tools for modelling
hybridization and mismatch-based interactions is the nearest neighbor method (NN)36 To explore
the strategy between the NN method and hybridization of DNA it is useful to understand some
details related to predicting the melting temperature (Tm)
First the system of interest will be defined at the equilibrium of dsDNA and ssDNA at the
point where both populations are equal ie half the strands of DNA are in the double helix state
and the other half are single-stranded and are often in various conformations Tm is the temperature
6
at which this equilibrium is found Next the equilibrium constant Keq is defined as being a ratio
of the concentration of dsDNA and ssDNA (as stated in Equation 1) A second expression can be
derived from the Vanrsquot Hoff equation (as stated in Equation 2) where ∆Ho and ∆So are the standard
enthalpy and entropy of hybridization and lnCT is the natural logarithm of the total strand
concentration This second equation can be used to calculate the thermodynamic parameters
related to Tm with the same being true vice versa37
Equation 1 = [][]
Equation 2 = ∆∆
With this foundation investigation into the NN method for modelling can be undertaken
The thermodynamics associated with a base pair are related to some degree with neighboring base
pairs Free energy values and other related parameters have been determined experimentally for
model oligonucleotide sequences This information is then used in conjunction with the nearest
neighbor algorithm (as presented in Equation 3) for obtaining the Tm for the strand of interest
Here base pair doublets are considered for sequence stability with ten unique combinations of
doublets (5rsquo-3rsquo) CG GC AT AA (also = TT) AG (also = CT) AC (also = GT) GA (also = TC)
GG (also = CC) TG (also = CA)38
Equation 3 ∆ = ∆ + ∆ + sum ∆
Equation 4 ∆ = ∆ minus ∆
In Equation 3 the ∆Gi(total) refers to the free energy of the strand of interest ∆G(init)
refers to the free energy of the strand of initiating base pair ∆G(sym) refers to the free energy of
symmetry Gj refers to the free energy associated with one of ten nearest neighbor stacking
interactions and nij is the appearance rate of the stacking interaction of interest Thermodynamic
parameters are also present for entropy and enthalpy allowing for the calculation of the Tm using
Equation 43638 Higher Tm values indicate greater stability than lower Tm values39ndash42 The NN
method can also be used along with a database of mismatch energetics to determine the
thermodynamic parameters related to those sequences
7
Tm values when used in conjunction with the free energies provide a theoretical basis for
designing probe ndash capture strand interactions This understanding can be useful when designing
wash conditions that control stringency for oligonucleotides composed of sequences with high
similarity Stringency control can be achieved using higher temperature (because increasing
temperature results in de-annealing of sequences and has greater effect on hybrids with partial
complementarity)43 by controlling the ionic strength of hybridization44 and via chaotropic agents
such as formamide45 and urea46 Although all strategies are valid for reducing false positive signals
(that arise from partially complementary strands of oligonucleotides) the use of washes containing
chaotropic agents may be more applicable for the POC given that temperature control requires a
temperature module
Chaotropic agents like formamide lower the melting temperature of duplex DNA by
engaging with the hydrogen bond network of DNA The degree by which temperature is lowered
depends on the GC content the conformations of single and duplex forms and the hydration state
of the strand (but typically can range 24 ndash 29 degC per mole of formamide)45 Chemically
formamide strongly associates with DNA is capable of four hydrogen bonds (same as water) and
is a stronger hydrogen bond acceptor than water Formamide ndash water bonds have been reported to
be 20 stronger than water ndash water bonds and it is accepted that formamide engages the hydration
network around DNA4547 Thus using formamide in washes for DNA hybridization can lower the
melting temperature favoring fully complementary hybrids over partially complementary hybrids
123 Notes and Considerations for POC Application
Developing a DNA screening device for the POC application requires consideration of the
many challenges faced by clinicians When screening genetic samples from blood it is important
to note that samples are often complex with proteins and other type of biomolecules (in addition
to cellular debris) and these materials may occlude the signal generated from target detection48
Another challenge to note for nucleic acid-based diagnostics is the low amount of target present in
clinical samples For example one milliliter of human blood contains approximately 107
leukocytes corresponding to femtomolar quantities (fM or attomoles 10-18 moles) of target nucleic
acid Thus detection strategies requiring hybridization-based assay require enzymatic
amplification of the target materials or improved analytical figures of merit for application in
POC49 Presently there are many different strategies for enzymatic amplification of nucleic acids
8
including polymerase chain reaction32 loop-mediated isothermal amplification34 helicase-
dependent isothermal amplification50 and recombinase polymerase amplification51 Post
amplification targets are often detected using hybridization-based assays using Watson-Crick base
pairing for detection of targets of interest Typically capture probes of complementary sequence
to targets are immobilized on a surface and the presence of target forms hybrids that are transduced
via electrochemical5253 or optical strategies54 Nano-surfaces can be integrated into this detection
strategy with oligonucleotides immobilized on the surface of nanoparticles allowing for
transduction via near-field phenomenon
13 Quantum dots
Nanomaterials based on gold and semiconductor composites have had a significant impact
across many different research fields including the chemical physical and biological sciences
Interest in nanoparticles has been driven due to the unique fundamental properties of these
materials as they approach and occupy size regions between bulk material and isolated atoms
Luminescent semiconductor-based quantum dots (QDs) in particular have attracted attention due
to their unique electro-optical properties arising from small size scales (typically ranging from
2 ndash 10 nm and consisting of 102 ndash 104 atoms per crystal) The key factors of interest for these
particles are material composition and size with a combination of the two giving rise to control of
physical properties such as the spectral profile and photon band gap energies55ndash59
There are many strategies for preparing and tuning the electro-optical properties of QDs
but some of the most studied from a synthetic perspective are based on binary composites of
elements from groups II-VI (like CdSe CdS or CdTe) and III-V (like InAs)55ndash58 For binary
composites luminescent properties can be controlled by choice of materials (selecting specific
regions of the UV-vis spectrum) and by control of size whereby smaller crystals are blue-shifted
and larger crystals are red-shifted5759 QDs used in most biological investigations are constructed
in a coreshell manner where the core is composed on a composite of materials previously
mentioned and the shell is composed of an inert coating (ie ZnS see Figure 2A) The QD shell
protects the nanoparticle from environmental degradation forming a protective layer and provides
a larger potential energy barrier for confining the exciton The shell material also provides a
synthetic strategy for controlling the core size and the type of shell allows for designing a class of
ligands for functionalizing the nanoparticle5556
9
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of
ZnS B Quantum dots of different colors are presented with their corresponding core size
image of solution and photoluminescence spectra and color C Diagram representing the
quantum confinement and the change in band gap energy as material size decreases below
the Bohr-exciton radius Here CB and VB represent the conduction and valence band
respectively and Eg represent the band gap energies Image adapted with permission
Copyright 2011 American Chemical Society60
The resulting particles have been incorporated into biological systems using surface ligands
with chemistry that allows the crystals to be stable in aqueous and physiological buffers6162
Further functionalization of these ligands has also allowed for the integration of biomolecules like
nucleic acids63 and proteins64 and polymers like polyethylene glycol (PEG) allowing applications
that extend from biological imaging65 to diagnostic device development and commercial
technologies566667 Optically quantum dots (QD) have broad absorption wavelengths (from the
UV into the visible) narrow and symmetrical emission photoluminescence (PL) profiles (25 nm
of full width at half maximum) high quantum yields and photochemical stability59 These
spectral properties in addition to the large surface area of QDs make them favourable donors for
RET processes
10
131 Quantum Confinement and The Particle in a Box
A brief overview of the quantum mechanics related to QDs will be discussed before
detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids
As the semiconducting material that composes QDs transitions from the bulk scale to the nano-
scale the valence and conductance bands of the semiconductor material split into discrete
energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the
composite of materials however for nanomaterials the band gap can also be tuned by modulating
the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the
dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like
CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an
electron-hole pair When an electron is excited by a photon of greater energy than the band gap
(the probability increases at higher energies yielding broad absorption spectra) the separation of
the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used
to describe this phenomenon is called quantum confinement and the model that best describes it is
the particle in a box575960
In this model a particle is said to be confined in a symmetrical box (of diameter a) where
the center of the box is denoted as = 0 and the edges of the box are denoted as = (
( Here
the potential energy inside the box +( le le
(- is said to be zero and the potential energy outside
the box + le ( ge
(- is said to be infinite The resulting probability of finding a particle outside
the confines of the box is zero 0 = 0 + le ( ge
(-1 and the discrete energy
eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of
interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material
The surface of the material represents the impenetrable barrier (potential energy is infinity)
restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869
As size of the QDs decreases the energy required for excitation increases because the
exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral
properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a
RET based system because the surface area of QDs allows for loading of multiple biomolecules
Thus multiple pathways of RET can take place that can collectively improve energy transfer
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
iii
Acknowledgments
I would like to begin by expressing my gratitude to Professor Ulrich J Krull for his guidance and
mentorship throughout my graduate career I am privileged to have worked in his lab with other
motivated and driven graduate students I have learned a lot at my time in the Chemical Sensors
Group (CSG) and I will always cherish my experience and memories here I am also grateful to
Professor Aaron R Wheeler who has graciously agreed to be the second reader for this thesis I
would also like to acknowledge Professor Paul A E Piunno with whom I have had many
conversations about research graduate work and judo Special thanks are also extended to Dr M
Omair Noor for his mentorship I have learned a lot about research from him that I would have
never have learned otherwise
I am grateful to numerous members of the University of Toronto Community for their help
Members of the UTM stores Microelectronics Academic workshop are sincerely thanked for their
support I would also like to thank the administrative staff at the UTM campus for their support
during my graduate work including Carmen Bryson Jessica Bailey Michelle Bae Christina M
Fortes and Roxana Moreira-Diaz
Much of the work in this thesis would not have been possible without the support of members from
the Department of Chemical and Physical Science and CSG I am forever grateful to Dr Abootaleb
Sedighi Dr Samer Doughan Dr Peter Mitrakos Dr Sreekumair Nair Dr Thottackad
Radhakrishnan Anna Shahmuradyan Yi Han Phillip Rolo Hifza Najib Muhammad Shahrukh
David Hrovat Richard Fuku Hamna Fayyaz and Alex Escobar for their support
Lastly I would like to acknowledge my family and friends for their continued support I would
like to express my gratitude to my sister brother-in-law and baby niece for always making life
enjoyable I would also like to thank my girlfriend for her continued support throughout my time
in grad school Finally none of this would have been possible without the sacrifice and
encouragement from my parents I am truly blessed to have you as my role models
iv
Table of Contents
Acknowledgments iii
Table of Contents iv
List of Tables vi
List of Figures vii
Chapter 1 1
Introduction 1
11 Cystic Fibrosis and Genes Associated with Cystic Fibrosis Transmembrane Protein 1
111 Nucleic Acids based Detection of Genes Associated with CF and ∆F508
mutation of CFTR Gene1
12 Nucleic Acids and Oligonucleotide Detection 3
121 Structure and Composition of DNA Hybridization 4
122 Thermodynamics of DNA Hybridization 5
123 Notes and Considerations for POC Application 7
13 Quantum dots 8
131 Quantum Confinement and The Particle in a Box 10
14 Fluorescence and Resonance Energy Transfer 11
141 Fluorescence Resonance Energy Transfer (FRET)11
15 Paper Based Analytical Devices 14
151 Paper Substrates for Sensing Technology Overview 15
152 Cellulose Modification and Smartphone-based Detection 15
16 Thesis Objectives and Contributions 17
Chapter 2 19
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation
Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera 19
21 Experimental 19
211 Methods20
v
212 Instrumentation 24
22 Results and Discussion 25
221 FRET Pair Characterization (gQD ndash Cy3) 25
222 Oligonucleotide Hybridization in Solution 26
223 Oligonucleotide Hybridization in Paper Substrates 28
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by
Smartphone Imaging 32
225 Analytical Figures of Merit 38
226 Selectivity for Mixtures of WT and MT Targets 40
227 Paper-based Assay Response for Complex Sample Matrices 42
228 Blind Assay for Detection and Quantification of CFTR Target Mixes 43
Chapter 3 45
Conclusion and Future Work 45
31 Future Directions 46
References 47
vi
List of Tables
Table 1 Summary of FDA approved Nucleic Acid Based Tests for Diagnosis of CF 2
Table 2 Oligonucleotide Sequences used in Hybridization Assays 20
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids 34
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids 34
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids 34
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids 34
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids 36
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids 36
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids 36
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids 37
Table 11 Analytical Performance Direct and Sandwich Bioassays 40
Table 12 Blind Assay for Direct and Sandwich Assays 44
vii
List of Figures
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and Crick
Arrows on the ribbons represent the directionality bias for the single strands and dimensions for
the polymer are presented with one turn of the helix every 34 nm the distance between base pairs
every 034 nm and the distance between the phosphate backbone and the central axis every 1 nm
B shows the hydrogen bonding taking place between complementary pairs of nucleobases as
proposed by Chargaff with adenine (A) having two hydrogen bonds with thymine (T) and guanine
(G) having three hydrogen bonds with cytosine (C) Image was adapted with permission
Copyright Nature Education 201331 5
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of ZnS
B Quantum dots of different colors are presented with their corresponding core size image of
solution and photoluminescence spectra and color C Diagram representing the quantum
confinement and the change in band gap energy as material size decreases below the Bohr-exciton
radius Here CB and VB represent the conduction and valence band respectively and Eg represent
the band gap energies Image adapted with permission Copyright 2011 American Chemical
Society60 9
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of colloidally
stable and spherical QD (green) with multiple FRET acceptors (yellow) (b) Change in FRET
efficiency based on changes in the distance between donor and acceptor (c) QD (green)
immobilized on a surface can interact with multiple FRET acceptors by interacting with adjacent
acceptors Image acquired with permission from Algar et al70 Copyright Elsevier 2010 12
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in blue)
are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3 on
the proximal end and upon hybridization is brought to proximity with gQDs allowing for FRET
to take place (B) In sandwich assay format the probe strand hybridizes with the target strand (seen
in red) such that there is an overhang on the distal end Reporter strand (seen in green) hybridizes
with the overhang region of the target strand bringing to proximity the Cy3 label on the proximal
end of the reporter 14
viii
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society of
Chemistry 2016 16
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A) Reaction
zones consisted of chemically modified paper that were conjugated with gQD-oligonucleotide
probes Zones contained WT and MT controls and test zones where unknown samples were
spotted and imaged Detection was based on the principle of RET with gQDs used as donors and
Cy3 labels on oligonucleotide strands as acceptors (B) Imaging used a smartphone camera with
data processing by ImageJ to split the image to RGB color channels 18
Figure 7 Image of buffer solution leakage from hydrophilic paper zones 23
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines and
emission is shown as solid lines 26
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by fluorescence
spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii) CFTR single
DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT target strands The
concentration-response curves for the different gQD-probe conjugates are shown A WT Cy3
labelled target strands are seen in blue and MT Cy3 labelled target strands are seen in orange
Normalized PL spectra for the calibration curves are shown for B) CFTR WT Cy3 labelled target
strands and C) CFTR MT Cy3 labelled target strands ( indicates increasing target concentration)
27
Figure 10 Representations of the two different direct assay formats investigated in solution phase
gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA MT probe
and were mixed with complementary CFTR WT Cy3 target strands and CFTR MT Cy3 target
strands Hybridization resulted in proximity of gQDs and Cy3 which resulted in FRET 28
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of Cy3
labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol (vii) 75
ix
pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of spots that
may not be visible otherwise 29
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers to
WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash MT target
(D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and Tm) were calculated
using the nearest neighbor method3839 30
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash MT target
(D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and Tm) were calculated
using the nearest neighbor method3839 32
Figure 14 Determination of optimal wash conditions for direct and sandwich assay considered
RG Ratios with variation of formamide concentration for wash times of 0 5 10 15 and 20 min
The optimal wash conditions for direct assay was found to be BB+10F for 5 minutes for (A)
gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal wash conditions for
sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-WT probe sequence and
BB+5F for 20 minutes for (D) gQD-MT probe sequence 38
Figure 15 Concentration-response curves showing the RG ratiometric response of the direct and
sandwich assay formats (Ai) gQD-WT probe conjugates were used for determination of Cy3
labelled WT targets and (Bi) gQD-MT probe conjugates were used for determination of Cy3
labelled MT targets (Ci) gQD-WT probe conjugates were used for determination of unlabelled
WT targets with Cy3 labelled reporters and (Di) gQD-MT probe conjugates were used for
determination of unlabelled MT targets with Cy3 labelled reporters The RG ratiometric response
of the direct assay at the low pmol concentration range was also determined (Aii) gQD-WT probe
conjugates and (Bii) gQD-MT probe conjugates The sandwich assay format (Cii) gQD-WT probe
conjugates and (Dii) gQD-MT probe conjugates Note that the scale for (A) and (B) is logarithmic
Each error bar represents one standard deviation for n=4 replicates 39
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes and
(Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined using
background corrected RG ratio plots for hybridization of gQD-probe conjugates with Cy3 labelled
x
targets (for direct assay A and B) and gQD-probe conjugates with unlabeled targets and Cy3
labelled reporter sequences (for sandwich assay C and D) Response of the hybridization assay
was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-wash (Bi and Di) MT
probe conjugates Post-wash assays yielded signal response shown in Aii and Cii for WT probe
conjugates and in Bii and Dii for MT probe conjugates Error bars represent one standard deviation
for n = 4 replicates 41
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates and
(B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to direct assay
and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was collected for (C)
gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars represent one standard
deviation for n = 4 replicates 42
1
Chapter 1
Introduction
11 Cystic Fibrosis and Genes Associated with Cystic Fibrosis Transmembrane Protein
Cystic fibrosis (CF) is a multi-system fatal autosomal recessive disorder that is
characterized by viscous secretions in the lungs of patients due to mutations in cystic fibrosis
transmembrane conductance regulator protein (CFTR) CF affects 1 in 3000 births with ~70000
people affected worldwide1ndash5 Over 1500 mutations for the CFTR protein have been found but few
are common and fewer result in the disease Of the few mutations responsible for the disease state
the deletion of phenylalanine at the 508 position (∆F508) is responsible for over two-thirds of the
cases while all other mutations account for no more than 5 of the cases individually256
Development of sensing technology for early detection of ∆F508 would serve to enable improved
screening by clinicians to identify the predominant gene carriers The strategies for diagnosing CF
are based on newborn screening (NBS) programs that work via screening for serum markers
including the immunoreactive trypsinogen (IRT) assay7ndash9 This assay is typically followed by
diagnosis of the genetic basis of disease including detection of ∆F508 and related mutations based
on determining the presence of specific oligonucleotide sequences Finally a sweat chloride test
is performed to diagnose patients with CF All of these techniques require skilled technicians to
process samples perform and analyse tests via resource-intensive technologies10 The aim of this
work is to contribute to the development of a low cost easy to use and portable method for sensing
CFTR ∆F508 gene mutations beginning with a focus on a suitable transduction strategy
111 Nucleic Acids based Detection of Genes Associated with CF and ∆F508 mutation of CFTR Gene
There are multiple strategies for transducing the presence of genes associated with CF and
some of the technologies that have been approved by the United Stated Food and Drug
Administration (FDA) for use as in-vitro medical devices are presented in Table 1 (accessed Feb
20th 2018)11
2
Table 1 Summary of FDA approved Nucleic Acid Based Tests for Diagnosis of CF
Manufacturer Trade Name Detection Strategy
Illumina Inc Illumina MiSeqDx Cystic
Fibrosis Clinical Sequencing
Assay
Next-gen sequencing by
synthesis
Illumina MiSeqDx Cystic
Fibrosis 139-Variant Assay
Luminex Molecular
Diagnostics Inc
xTAG Cystic Fibrosis 60 kit v2 Microbead-dye barcode
coupled microarray analysis xTAG Cystic Fibrosis 39 kit v2
Osmetech Molecular
Diagnostics
eSensor CF Genotyping Test Sandwich hybridization assay
with ferrocene tag for cyclic
voltammetry analysis
Nanosphere Inc Verigene CFTR and Verigene
CFTR PolyT Nucleic Acid Tests
Genomic amplification
followed by sandwich assay
with probes and gold
nanoparticle reporters for
analysis
Third Wave Technology Inc InPlex CF Molecular Test PCR coupled with FRET based
microwell plate
Celera Diagnostics Cystic Fibrosis Genotyping
Assay
PCR coupled with capillary
electrophoresis and
oligonucleotide ligation assay
Typically these technologies require the use of specialized facilities and dedicated
technicians for analysis of patient samples and confirmation of CF may take up to a few weeks79
The resources and time required to diagnose patients may be reduced through the development of
point-of-care (POC) devices In particular the use of paper-based test strips with smartphone
detection for on-site rapid screening of disease markers would serve to alleviate the burden placed
on the health care system by more expensive techniques12
At the core of POC technology is the transduction strategy and much effort has gone into
developing optical13 and electrochemical methods14 for generating and measuring signal Yet the
application of this technology has not been investigated for selective sensing of similar nucleic
acid sequences that are often found to be associated with the genetic basis of disease Thus to
further discuss the challenges in this field it is important to address some of the background
technology that has been developed for POC sensors In particular this chapter will discuss nucleic
acid detection and the thermodynamics associated with hybridization interactions the use of
3
formamide as a chaotrope for controlling hybridization the use of nanomaterials like quantum dots
as integrated components in the bioassays for fluorescence resonance energy transfer-based
sensing strategies and the application of paper as a platform and substrate for sensing
12 Nucleic Acids and Oligonucleotide Detection
Deoxyribonucleic acid (DNA) is a class of biopolymers that stores hereditary information
and ribonucleic acid (RNA) functions as the set of instructions for synthesis of proteins15 The two-
step process by which the DNA nucleobase sequence is transcribed for production of RNA and
subsequently RNA is used as a template for translation to produce proteins is referred to as the
central dogma of molecular biology16 Proteins carry out the function that is encoded in the genetic
regions of DNA by interacting with other molecules and biopolymers present within and on the
surface of cells The specific interactions that govern the proteinsrsquo function are due to the three-
dimensional structure of the amino acid sequence that composes proteins17 The order of amino
acids which composes proteins is based on the nucleobase sequence of transcribing RNA (and
therefore DNA) Thus hereditary information stored as the base sequence of DNA can govern the
sequence of amino acids and therefore the structure and function of proteins1617 There are
numerous types of diseases that have arisen due to nucleobase-pair mutations in the sequence of
gene coding regions of DNA18 Mutations of DNA bases influence the amino acid sequence that
compose proteins and a three-base pair deletion like the one found for phenylalanine at codon 508
significantly alters the function of the protein associated with the CFTR gene Other types of
genetic diseases also arise due to mutations of the base pair sequence associated with DNA and
strategies for detection of nucleic acid mutations offer a method to detect the presence of a disease
state
To determine the genetic basis of disease for guiding clinical treatment diagnostic
technology for sensing nucleic acids must be further developed The main goal of clinical
diagnostic technology is to determine the molecular basis of disease for guiding patient therapy
because knowledge obtained from diagnostics are paramount for programing treatment strategies
Clinical diagnostics using a POC strategy offers improved opportunity for wide-scale screening
due to the advantages of low cost ease of manufacturing ease of transport ease of use and ease
of disposal19 One approach to the detection of genetic materials (deoxyribonucleic acid) is based
4
on hybridization and this process requires consideration of the chemical composition structure
and thermodynamics associated with hybridization
121 Structure and Composition of DNA Hybridization
Elucidation of DNArsquos structure and function has a long-storied history that has impacted
many fields of research including chemistry biology and medicine Much of the early work
related to DNA was focused on the structure of DNA with scientists focusing on the key details
related to the chemical composition of the monomers and the structural format of the polymeric
structure of DNA20ndash24 The key findings of the structure of DNA are summarized as follows
1 The structure for the DNA salt is composed of two helical polymer chains that are
coiled around one another and around a shared axis (see Figure 1A) The outside of the
chains is composed of phosphate-sugars groups and the chains are linked together on
the inside via hydrogen bonds between the nucleotide bases
2 The chains are anti-parallel in conformation with ie the 5rsquo end of one chain is bound
via the nucleobases to the 3rsquo end of the other chain
3 Both chains follow a right-handed helix (one type of DNA known as Z-DNA follows
a left-handed helix but this was discovered later)25 and base residues are present on the
chains every 34 Å with structural repeats every ten residues The distance from the
central shared axis to the phosphorous atom is 10 Å
4 The four bases composing DNA bond to a specific pair (see Figure 1B) ie adenine
(purine) binds with thymine (pyrimidine) and guanine (purine) binds with cytosine
(pyrimidine) The relationship of molar equivalency between pairs of bases ie A ndash T
and G ndash C was determined earlier by Chargaff in 195026
Details related to the structure and composition of DNA has formed the basis of our
understanding of the role of DNA in molecular and cell biology Through the structure of DNA
the mechanism for DNA replication27 transcription28 and translation29 for protein synthesis was
elucidated The confirmation of DNA as the storage for hereditary information paved the way for
initiatives like the Human Genome Project and insights from this undertaking have fueled research
regarding the genetic basis of disease30
5
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and
Crick Arrows on the ribbons represent the directionality bias for the single strands and
dimensions for the polymer are presented with one turn of the helix every 34 nm the
distance between base pairs every 034 nm and the distance between the phosphate
backbone and the central axis every 1 nm B shows the hydrogen bonding taking place
between complementary pairs of nucleobases as proposed by Chargaff with adenine (A)
having two hydrogen bonds with thymine (T) and guanine (G) having three hydrogen bonds
with cytosine (C) Image was adapted with permission Copyright Nature Education 201331
122 Thermodynamics of DNA Hybridization
Design and development of DNA-based technologies have been guided by the
thermodynamic modelling of hybridization Techniques like PCR3233 and isothermal
amplification34 rely on accurate control over the annealing of primers and DNA sensors often uses
temperature and chaotropic agents for achieving selectivity35 One of the useful tools for modelling
hybridization and mismatch-based interactions is the nearest neighbor method (NN)36 To explore
the strategy between the NN method and hybridization of DNA it is useful to understand some
details related to predicting the melting temperature (Tm)
First the system of interest will be defined at the equilibrium of dsDNA and ssDNA at the
point where both populations are equal ie half the strands of DNA are in the double helix state
and the other half are single-stranded and are often in various conformations Tm is the temperature
6
at which this equilibrium is found Next the equilibrium constant Keq is defined as being a ratio
of the concentration of dsDNA and ssDNA (as stated in Equation 1) A second expression can be
derived from the Vanrsquot Hoff equation (as stated in Equation 2) where ∆Ho and ∆So are the standard
enthalpy and entropy of hybridization and lnCT is the natural logarithm of the total strand
concentration This second equation can be used to calculate the thermodynamic parameters
related to Tm with the same being true vice versa37
Equation 1 = [][]
Equation 2 = ∆∆
With this foundation investigation into the NN method for modelling can be undertaken
The thermodynamics associated with a base pair are related to some degree with neighboring base
pairs Free energy values and other related parameters have been determined experimentally for
model oligonucleotide sequences This information is then used in conjunction with the nearest
neighbor algorithm (as presented in Equation 3) for obtaining the Tm for the strand of interest
Here base pair doublets are considered for sequence stability with ten unique combinations of
doublets (5rsquo-3rsquo) CG GC AT AA (also = TT) AG (also = CT) AC (also = GT) GA (also = TC)
GG (also = CC) TG (also = CA)38
Equation 3 ∆ = ∆ + ∆ + sum ∆
Equation 4 ∆ = ∆ minus ∆
In Equation 3 the ∆Gi(total) refers to the free energy of the strand of interest ∆G(init)
refers to the free energy of the strand of initiating base pair ∆G(sym) refers to the free energy of
symmetry Gj refers to the free energy associated with one of ten nearest neighbor stacking
interactions and nij is the appearance rate of the stacking interaction of interest Thermodynamic
parameters are also present for entropy and enthalpy allowing for the calculation of the Tm using
Equation 43638 Higher Tm values indicate greater stability than lower Tm values39ndash42 The NN
method can also be used along with a database of mismatch energetics to determine the
thermodynamic parameters related to those sequences
7
Tm values when used in conjunction with the free energies provide a theoretical basis for
designing probe ndash capture strand interactions This understanding can be useful when designing
wash conditions that control stringency for oligonucleotides composed of sequences with high
similarity Stringency control can be achieved using higher temperature (because increasing
temperature results in de-annealing of sequences and has greater effect on hybrids with partial
complementarity)43 by controlling the ionic strength of hybridization44 and via chaotropic agents
such as formamide45 and urea46 Although all strategies are valid for reducing false positive signals
(that arise from partially complementary strands of oligonucleotides) the use of washes containing
chaotropic agents may be more applicable for the POC given that temperature control requires a
temperature module
Chaotropic agents like formamide lower the melting temperature of duplex DNA by
engaging with the hydrogen bond network of DNA The degree by which temperature is lowered
depends on the GC content the conformations of single and duplex forms and the hydration state
of the strand (but typically can range 24 ndash 29 degC per mole of formamide)45 Chemically
formamide strongly associates with DNA is capable of four hydrogen bonds (same as water) and
is a stronger hydrogen bond acceptor than water Formamide ndash water bonds have been reported to
be 20 stronger than water ndash water bonds and it is accepted that formamide engages the hydration
network around DNA4547 Thus using formamide in washes for DNA hybridization can lower the
melting temperature favoring fully complementary hybrids over partially complementary hybrids
123 Notes and Considerations for POC Application
Developing a DNA screening device for the POC application requires consideration of the
many challenges faced by clinicians When screening genetic samples from blood it is important
to note that samples are often complex with proteins and other type of biomolecules (in addition
to cellular debris) and these materials may occlude the signal generated from target detection48
Another challenge to note for nucleic acid-based diagnostics is the low amount of target present in
clinical samples For example one milliliter of human blood contains approximately 107
leukocytes corresponding to femtomolar quantities (fM or attomoles 10-18 moles) of target nucleic
acid Thus detection strategies requiring hybridization-based assay require enzymatic
amplification of the target materials or improved analytical figures of merit for application in
POC49 Presently there are many different strategies for enzymatic amplification of nucleic acids
8
including polymerase chain reaction32 loop-mediated isothermal amplification34 helicase-
dependent isothermal amplification50 and recombinase polymerase amplification51 Post
amplification targets are often detected using hybridization-based assays using Watson-Crick base
pairing for detection of targets of interest Typically capture probes of complementary sequence
to targets are immobilized on a surface and the presence of target forms hybrids that are transduced
via electrochemical5253 or optical strategies54 Nano-surfaces can be integrated into this detection
strategy with oligonucleotides immobilized on the surface of nanoparticles allowing for
transduction via near-field phenomenon
13 Quantum dots
Nanomaterials based on gold and semiconductor composites have had a significant impact
across many different research fields including the chemical physical and biological sciences
Interest in nanoparticles has been driven due to the unique fundamental properties of these
materials as they approach and occupy size regions between bulk material and isolated atoms
Luminescent semiconductor-based quantum dots (QDs) in particular have attracted attention due
to their unique electro-optical properties arising from small size scales (typically ranging from
2 ndash 10 nm and consisting of 102 ndash 104 atoms per crystal) The key factors of interest for these
particles are material composition and size with a combination of the two giving rise to control of
physical properties such as the spectral profile and photon band gap energies55ndash59
There are many strategies for preparing and tuning the electro-optical properties of QDs
but some of the most studied from a synthetic perspective are based on binary composites of
elements from groups II-VI (like CdSe CdS or CdTe) and III-V (like InAs)55ndash58 For binary
composites luminescent properties can be controlled by choice of materials (selecting specific
regions of the UV-vis spectrum) and by control of size whereby smaller crystals are blue-shifted
and larger crystals are red-shifted5759 QDs used in most biological investigations are constructed
in a coreshell manner where the core is composed on a composite of materials previously
mentioned and the shell is composed of an inert coating (ie ZnS see Figure 2A) The QD shell
protects the nanoparticle from environmental degradation forming a protective layer and provides
a larger potential energy barrier for confining the exciton The shell material also provides a
synthetic strategy for controlling the core size and the type of shell allows for designing a class of
ligands for functionalizing the nanoparticle5556
9
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of
ZnS B Quantum dots of different colors are presented with their corresponding core size
image of solution and photoluminescence spectra and color C Diagram representing the
quantum confinement and the change in band gap energy as material size decreases below
the Bohr-exciton radius Here CB and VB represent the conduction and valence band
respectively and Eg represent the band gap energies Image adapted with permission
Copyright 2011 American Chemical Society60
The resulting particles have been incorporated into biological systems using surface ligands
with chemistry that allows the crystals to be stable in aqueous and physiological buffers6162
Further functionalization of these ligands has also allowed for the integration of biomolecules like
nucleic acids63 and proteins64 and polymers like polyethylene glycol (PEG) allowing applications
that extend from biological imaging65 to diagnostic device development and commercial
technologies566667 Optically quantum dots (QD) have broad absorption wavelengths (from the
UV into the visible) narrow and symmetrical emission photoluminescence (PL) profiles (25 nm
of full width at half maximum) high quantum yields and photochemical stability59 These
spectral properties in addition to the large surface area of QDs make them favourable donors for
RET processes
10
131 Quantum Confinement and The Particle in a Box
A brief overview of the quantum mechanics related to QDs will be discussed before
detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids
As the semiconducting material that composes QDs transitions from the bulk scale to the nano-
scale the valence and conductance bands of the semiconductor material split into discrete
energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the
composite of materials however for nanomaterials the band gap can also be tuned by modulating
the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the
dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like
CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an
electron-hole pair When an electron is excited by a photon of greater energy than the band gap
(the probability increases at higher energies yielding broad absorption spectra) the separation of
the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used
to describe this phenomenon is called quantum confinement and the model that best describes it is
the particle in a box575960
In this model a particle is said to be confined in a symmetrical box (of diameter a) where
the center of the box is denoted as = 0 and the edges of the box are denoted as = (
( Here
the potential energy inside the box +( le le
(- is said to be zero and the potential energy outside
the box + le ( ge
(- is said to be infinite The resulting probability of finding a particle outside
the confines of the box is zero 0 = 0 + le ( ge
(-1 and the discrete energy
eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of
interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material
The surface of the material represents the impenetrable barrier (potential energy is infinity)
restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869
As size of the QDs decreases the energy required for excitation increases because the
exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral
properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a
RET based system because the surface area of QDs allows for loading of multiple biomolecules
Thus multiple pathways of RET can take place that can collectively improve energy transfer
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
iv
Table of Contents
Acknowledgments iii
Table of Contents iv
List of Tables vi
List of Figures vii
Chapter 1 1
Introduction 1
11 Cystic Fibrosis and Genes Associated with Cystic Fibrosis Transmembrane Protein 1
111 Nucleic Acids based Detection of Genes Associated with CF and ∆F508
mutation of CFTR Gene1
12 Nucleic Acids and Oligonucleotide Detection 3
121 Structure and Composition of DNA Hybridization 4
122 Thermodynamics of DNA Hybridization 5
123 Notes and Considerations for POC Application 7
13 Quantum dots 8
131 Quantum Confinement and The Particle in a Box 10
14 Fluorescence and Resonance Energy Transfer 11
141 Fluorescence Resonance Energy Transfer (FRET)11
15 Paper Based Analytical Devices 14
151 Paper Substrates for Sensing Technology Overview 15
152 Cellulose Modification and Smartphone-based Detection 15
16 Thesis Objectives and Contributions 17
Chapter 2 19
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation
Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera 19
21 Experimental 19
211 Methods20
v
212 Instrumentation 24
22 Results and Discussion 25
221 FRET Pair Characterization (gQD ndash Cy3) 25
222 Oligonucleotide Hybridization in Solution 26
223 Oligonucleotide Hybridization in Paper Substrates 28
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by
Smartphone Imaging 32
225 Analytical Figures of Merit 38
226 Selectivity for Mixtures of WT and MT Targets 40
227 Paper-based Assay Response for Complex Sample Matrices 42
228 Blind Assay for Detection and Quantification of CFTR Target Mixes 43
Chapter 3 45
Conclusion and Future Work 45
31 Future Directions 46
References 47
vi
List of Tables
Table 1 Summary of FDA approved Nucleic Acid Based Tests for Diagnosis of CF 2
Table 2 Oligonucleotide Sequences used in Hybridization Assays 20
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids 34
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids 34
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids 34
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids 34
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids 36
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids 36
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids 36
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids 37
Table 11 Analytical Performance Direct and Sandwich Bioassays 40
Table 12 Blind Assay for Direct and Sandwich Assays 44
vii
List of Figures
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and Crick
Arrows on the ribbons represent the directionality bias for the single strands and dimensions for
the polymer are presented with one turn of the helix every 34 nm the distance between base pairs
every 034 nm and the distance between the phosphate backbone and the central axis every 1 nm
B shows the hydrogen bonding taking place between complementary pairs of nucleobases as
proposed by Chargaff with adenine (A) having two hydrogen bonds with thymine (T) and guanine
(G) having three hydrogen bonds with cytosine (C) Image was adapted with permission
Copyright Nature Education 201331 5
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of ZnS
B Quantum dots of different colors are presented with their corresponding core size image of
solution and photoluminescence spectra and color C Diagram representing the quantum
confinement and the change in band gap energy as material size decreases below the Bohr-exciton
radius Here CB and VB represent the conduction and valence band respectively and Eg represent
the band gap energies Image adapted with permission Copyright 2011 American Chemical
Society60 9
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of colloidally
stable and spherical QD (green) with multiple FRET acceptors (yellow) (b) Change in FRET
efficiency based on changes in the distance between donor and acceptor (c) QD (green)
immobilized on a surface can interact with multiple FRET acceptors by interacting with adjacent
acceptors Image acquired with permission from Algar et al70 Copyright Elsevier 2010 12
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in blue)
are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3 on
the proximal end and upon hybridization is brought to proximity with gQDs allowing for FRET
to take place (B) In sandwich assay format the probe strand hybridizes with the target strand (seen
in red) such that there is an overhang on the distal end Reporter strand (seen in green) hybridizes
with the overhang region of the target strand bringing to proximity the Cy3 label on the proximal
end of the reporter 14
viii
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society of
Chemistry 2016 16
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A) Reaction
zones consisted of chemically modified paper that were conjugated with gQD-oligonucleotide
probes Zones contained WT and MT controls and test zones where unknown samples were
spotted and imaged Detection was based on the principle of RET with gQDs used as donors and
Cy3 labels on oligonucleotide strands as acceptors (B) Imaging used a smartphone camera with
data processing by ImageJ to split the image to RGB color channels 18
Figure 7 Image of buffer solution leakage from hydrophilic paper zones 23
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines and
emission is shown as solid lines 26
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by fluorescence
spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii) CFTR single
DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT target strands The
concentration-response curves for the different gQD-probe conjugates are shown A WT Cy3
labelled target strands are seen in blue and MT Cy3 labelled target strands are seen in orange
Normalized PL spectra for the calibration curves are shown for B) CFTR WT Cy3 labelled target
strands and C) CFTR MT Cy3 labelled target strands ( indicates increasing target concentration)
27
Figure 10 Representations of the two different direct assay formats investigated in solution phase
gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA MT probe
and were mixed with complementary CFTR WT Cy3 target strands and CFTR MT Cy3 target
strands Hybridization resulted in proximity of gQDs and Cy3 which resulted in FRET 28
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of Cy3
labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol (vii) 75
ix
pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of spots that
may not be visible otherwise 29
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers to
WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash MT target
(D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and Tm) were calculated
using the nearest neighbor method3839 30
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash MT target
(D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and Tm) were calculated
using the nearest neighbor method3839 32
Figure 14 Determination of optimal wash conditions for direct and sandwich assay considered
RG Ratios with variation of formamide concentration for wash times of 0 5 10 15 and 20 min
The optimal wash conditions for direct assay was found to be BB+10F for 5 minutes for (A)
gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal wash conditions for
sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-WT probe sequence and
BB+5F for 20 minutes for (D) gQD-MT probe sequence 38
Figure 15 Concentration-response curves showing the RG ratiometric response of the direct and
sandwich assay formats (Ai) gQD-WT probe conjugates were used for determination of Cy3
labelled WT targets and (Bi) gQD-MT probe conjugates were used for determination of Cy3
labelled MT targets (Ci) gQD-WT probe conjugates were used for determination of unlabelled
WT targets with Cy3 labelled reporters and (Di) gQD-MT probe conjugates were used for
determination of unlabelled MT targets with Cy3 labelled reporters The RG ratiometric response
of the direct assay at the low pmol concentration range was also determined (Aii) gQD-WT probe
conjugates and (Bii) gQD-MT probe conjugates The sandwich assay format (Cii) gQD-WT probe
conjugates and (Dii) gQD-MT probe conjugates Note that the scale for (A) and (B) is logarithmic
Each error bar represents one standard deviation for n=4 replicates 39
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes and
(Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined using
background corrected RG ratio plots for hybridization of gQD-probe conjugates with Cy3 labelled
x
targets (for direct assay A and B) and gQD-probe conjugates with unlabeled targets and Cy3
labelled reporter sequences (for sandwich assay C and D) Response of the hybridization assay
was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-wash (Bi and Di) MT
probe conjugates Post-wash assays yielded signal response shown in Aii and Cii for WT probe
conjugates and in Bii and Dii for MT probe conjugates Error bars represent one standard deviation
for n = 4 replicates 41
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates and
(B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to direct assay
and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was collected for (C)
gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars represent one standard
deviation for n = 4 replicates 42
1
Chapter 1
Introduction
11 Cystic Fibrosis and Genes Associated with Cystic Fibrosis Transmembrane Protein
Cystic fibrosis (CF) is a multi-system fatal autosomal recessive disorder that is
characterized by viscous secretions in the lungs of patients due to mutations in cystic fibrosis
transmembrane conductance regulator protein (CFTR) CF affects 1 in 3000 births with ~70000
people affected worldwide1ndash5 Over 1500 mutations for the CFTR protein have been found but few
are common and fewer result in the disease Of the few mutations responsible for the disease state
the deletion of phenylalanine at the 508 position (∆F508) is responsible for over two-thirds of the
cases while all other mutations account for no more than 5 of the cases individually256
Development of sensing technology for early detection of ∆F508 would serve to enable improved
screening by clinicians to identify the predominant gene carriers The strategies for diagnosing CF
are based on newborn screening (NBS) programs that work via screening for serum markers
including the immunoreactive trypsinogen (IRT) assay7ndash9 This assay is typically followed by
diagnosis of the genetic basis of disease including detection of ∆F508 and related mutations based
on determining the presence of specific oligonucleotide sequences Finally a sweat chloride test
is performed to diagnose patients with CF All of these techniques require skilled technicians to
process samples perform and analyse tests via resource-intensive technologies10 The aim of this
work is to contribute to the development of a low cost easy to use and portable method for sensing
CFTR ∆F508 gene mutations beginning with a focus on a suitable transduction strategy
111 Nucleic Acids based Detection of Genes Associated with CF and ∆F508 mutation of CFTR Gene
There are multiple strategies for transducing the presence of genes associated with CF and
some of the technologies that have been approved by the United Stated Food and Drug
Administration (FDA) for use as in-vitro medical devices are presented in Table 1 (accessed Feb
20th 2018)11
2
Table 1 Summary of FDA approved Nucleic Acid Based Tests for Diagnosis of CF
Manufacturer Trade Name Detection Strategy
Illumina Inc Illumina MiSeqDx Cystic
Fibrosis Clinical Sequencing
Assay
Next-gen sequencing by
synthesis
Illumina MiSeqDx Cystic
Fibrosis 139-Variant Assay
Luminex Molecular
Diagnostics Inc
xTAG Cystic Fibrosis 60 kit v2 Microbead-dye barcode
coupled microarray analysis xTAG Cystic Fibrosis 39 kit v2
Osmetech Molecular
Diagnostics
eSensor CF Genotyping Test Sandwich hybridization assay
with ferrocene tag for cyclic
voltammetry analysis
Nanosphere Inc Verigene CFTR and Verigene
CFTR PolyT Nucleic Acid Tests
Genomic amplification
followed by sandwich assay
with probes and gold
nanoparticle reporters for
analysis
Third Wave Technology Inc InPlex CF Molecular Test PCR coupled with FRET based
microwell plate
Celera Diagnostics Cystic Fibrosis Genotyping
Assay
PCR coupled with capillary
electrophoresis and
oligonucleotide ligation assay
Typically these technologies require the use of specialized facilities and dedicated
technicians for analysis of patient samples and confirmation of CF may take up to a few weeks79
The resources and time required to diagnose patients may be reduced through the development of
point-of-care (POC) devices In particular the use of paper-based test strips with smartphone
detection for on-site rapid screening of disease markers would serve to alleviate the burden placed
on the health care system by more expensive techniques12
At the core of POC technology is the transduction strategy and much effort has gone into
developing optical13 and electrochemical methods14 for generating and measuring signal Yet the
application of this technology has not been investigated for selective sensing of similar nucleic
acid sequences that are often found to be associated with the genetic basis of disease Thus to
further discuss the challenges in this field it is important to address some of the background
technology that has been developed for POC sensors In particular this chapter will discuss nucleic
acid detection and the thermodynamics associated with hybridization interactions the use of
3
formamide as a chaotrope for controlling hybridization the use of nanomaterials like quantum dots
as integrated components in the bioassays for fluorescence resonance energy transfer-based
sensing strategies and the application of paper as a platform and substrate for sensing
12 Nucleic Acids and Oligonucleotide Detection
Deoxyribonucleic acid (DNA) is a class of biopolymers that stores hereditary information
and ribonucleic acid (RNA) functions as the set of instructions for synthesis of proteins15 The two-
step process by which the DNA nucleobase sequence is transcribed for production of RNA and
subsequently RNA is used as a template for translation to produce proteins is referred to as the
central dogma of molecular biology16 Proteins carry out the function that is encoded in the genetic
regions of DNA by interacting with other molecules and biopolymers present within and on the
surface of cells The specific interactions that govern the proteinsrsquo function are due to the three-
dimensional structure of the amino acid sequence that composes proteins17 The order of amino
acids which composes proteins is based on the nucleobase sequence of transcribing RNA (and
therefore DNA) Thus hereditary information stored as the base sequence of DNA can govern the
sequence of amino acids and therefore the structure and function of proteins1617 There are
numerous types of diseases that have arisen due to nucleobase-pair mutations in the sequence of
gene coding regions of DNA18 Mutations of DNA bases influence the amino acid sequence that
compose proteins and a three-base pair deletion like the one found for phenylalanine at codon 508
significantly alters the function of the protein associated with the CFTR gene Other types of
genetic diseases also arise due to mutations of the base pair sequence associated with DNA and
strategies for detection of nucleic acid mutations offer a method to detect the presence of a disease
state
To determine the genetic basis of disease for guiding clinical treatment diagnostic
technology for sensing nucleic acids must be further developed The main goal of clinical
diagnostic technology is to determine the molecular basis of disease for guiding patient therapy
because knowledge obtained from diagnostics are paramount for programing treatment strategies
Clinical diagnostics using a POC strategy offers improved opportunity for wide-scale screening
due to the advantages of low cost ease of manufacturing ease of transport ease of use and ease
of disposal19 One approach to the detection of genetic materials (deoxyribonucleic acid) is based
4
on hybridization and this process requires consideration of the chemical composition structure
and thermodynamics associated with hybridization
121 Structure and Composition of DNA Hybridization
Elucidation of DNArsquos structure and function has a long-storied history that has impacted
many fields of research including chemistry biology and medicine Much of the early work
related to DNA was focused on the structure of DNA with scientists focusing on the key details
related to the chemical composition of the monomers and the structural format of the polymeric
structure of DNA20ndash24 The key findings of the structure of DNA are summarized as follows
1 The structure for the DNA salt is composed of two helical polymer chains that are
coiled around one another and around a shared axis (see Figure 1A) The outside of the
chains is composed of phosphate-sugars groups and the chains are linked together on
the inside via hydrogen bonds between the nucleotide bases
2 The chains are anti-parallel in conformation with ie the 5rsquo end of one chain is bound
via the nucleobases to the 3rsquo end of the other chain
3 Both chains follow a right-handed helix (one type of DNA known as Z-DNA follows
a left-handed helix but this was discovered later)25 and base residues are present on the
chains every 34 Å with structural repeats every ten residues The distance from the
central shared axis to the phosphorous atom is 10 Å
4 The four bases composing DNA bond to a specific pair (see Figure 1B) ie adenine
(purine) binds with thymine (pyrimidine) and guanine (purine) binds with cytosine
(pyrimidine) The relationship of molar equivalency between pairs of bases ie A ndash T
and G ndash C was determined earlier by Chargaff in 195026
Details related to the structure and composition of DNA has formed the basis of our
understanding of the role of DNA in molecular and cell biology Through the structure of DNA
the mechanism for DNA replication27 transcription28 and translation29 for protein synthesis was
elucidated The confirmation of DNA as the storage for hereditary information paved the way for
initiatives like the Human Genome Project and insights from this undertaking have fueled research
regarding the genetic basis of disease30
5
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and
Crick Arrows on the ribbons represent the directionality bias for the single strands and
dimensions for the polymer are presented with one turn of the helix every 34 nm the
distance between base pairs every 034 nm and the distance between the phosphate
backbone and the central axis every 1 nm B shows the hydrogen bonding taking place
between complementary pairs of nucleobases as proposed by Chargaff with adenine (A)
having two hydrogen bonds with thymine (T) and guanine (G) having three hydrogen bonds
with cytosine (C) Image was adapted with permission Copyright Nature Education 201331
122 Thermodynamics of DNA Hybridization
Design and development of DNA-based technologies have been guided by the
thermodynamic modelling of hybridization Techniques like PCR3233 and isothermal
amplification34 rely on accurate control over the annealing of primers and DNA sensors often uses
temperature and chaotropic agents for achieving selectivity35 One of the useful tools for modelling
hybridization and mismatch-based interactions is the nearest neighbor method (NN)36 To explore
the strategy between the NN method and hybridization of DNA it is useful to understand some
details related to predicting the melting temperature (Tm)
First the system of interest will be defined at the equilibrium of dsDNA and ssDNA at the
point where both populations are equal ie half the strands of DNA are in the double helix state
and the other half are single-stranded and are often in various conformations Tm is the temperature
6
at which this equilibrium is found Next the equilibrium constant Keq is defined as being a ratio
of the concentration of dsDNA and ssDNA (as stated in Equation 1) A second expression can be
derived from the Vanrsquot Hoff equation (as stated in Equation 2) where ∆Ho and ∆So are the standard
enthalpy and entropy of hybridization and lnCT is the natural logarithm of the total strand
concentration This second equation can be used to calculate the thermodynamic parameters
related to Tm with the same being true vice versa37
Equation 1 = [][]
Equation 2 = ∆∆
With this foundation investigation into the NN method for modelling can be undertaken
The thermodynamics associated with a base pair are related to some degree with neighboring base
pairs Free energy values and other related parameters have been determined experimentally for
model oligonucleotide sequences This information is then used in conjunction with the nearest
neighbor algorithm (as presented in Equation 3) for obtaining the Tm for the strand of interest
Here base pair doublets are considered for sequence stability with ten unique combinations of
doublets (5rsquo-3rsquo) CG GC AT AA (also = TT) AG (also = CT) AC (also = GT) GA (also = TC)
GG (also = CC) TG (also = CA)38
Equation 3 ∆ = ∆ + ∆ + sum ∆
Equation 4 ∆ = ∆ minus ∆
In Equation 3 the ∆Gi(total) refers to the free energy of the strand of interest ∆G(init)
refers to the free energy of the strand of initiating base pair ∆G(sym) refers to the free energy of
symmetry Gj refers to the free energy associated with one of ten nearest neighbor stacking
interactions and nij is the appearance rate of the stacking interaction of interest Thermodynamic
parameters are also present for entropy and enthalpy allowing for the calculation of the Tm using
Equation 43638 Higher Tm values indicate greater stability than lower Tm values39ndash42 The NN
method can also be used along with a database of mismatch energetics to determine the
thermodynamic parameters related to those sequences
7
Tm values when used in conjunction with the free energies provide a theoretical basis for
designing probe ndash capture strand interactions This understanding can be useful when designing
wash conditions that control stringency for oligonucleotides composed of sequences with high
similarity Stringency control can be achieved using higher temperature (because increasing
temperature results in de-annealing of sequences and has greater effect on hybrids with partial
complementarity)43 by controlling the ionic strength of hybridization44 and via chaotropic agents
such as formamide45 and urea46 Although all strategies are valid for reducing false positive signals
(that arise from partially complementary strands of oligonucleotides) the use of washes containing
chaotropic agents may be more applicable for the POC given that temperature control requires a
temperature module
Chaotropic agents like formamide lower the melting temperature of duplex DNA by
engaging with the hydrogen bond network of DNA The degree by which temperature is lowered
depends on the GC content the conformations of single and duplex forms and the hydration state
of the strand (but typically can range 24 ndash 29 degC per mole of formamide)45 Chemically
formamide strongly associates with DNA is capable of four hydrogen bonds (same as water) and
is a stronger hydrogen bond acceptor than water Formamide ndash water bonds have been reported to
be 20 stronger than water ndash water bonds and it is accepted that formamide engages the hydration
network around DNA4547 Thus using formamide in washes for DNA hybridization can lower the
melting temperature favoring fully complementary hybrids over partially complementary hybrids
123 Notes and Considerations for POC Application
Developing a DNA screening device for the POC application requires consideration of the
many challenges faced by clinicians When screening genetic samples from blood it is important
to note that samples are often complex with proteins and other type of biomolecules (in addition
to cellular debris) and these materials may occlude the signal generated from target detection48
Another challenge to note for nucleic acid-based diagnostics is the low amount of target present in
clinical samples For example one milliliter of human blood contains approximately 107
leukocytes corresponding to femtomolar quantities (fM or attomoles 10-18 moles) of target nucleic
acid Thus detection strategies requiring hybridization-based assay require enzymatic
amplification of the target materials or improved analytical figures of merit for application in
POC49 Presently there are many different strategies for enzymatic amplification of nucleic acids
8
including polymerase chain reaction32 loop-mediated isothermal amplification34 helicase-
dependent isothermal amplification50 and recombinase polymerase amplification51 Post
amplification targets are often detected using hybridization-based assays using Watson-Crick base
pairing for detection of targets of interest Typically capture probes of complementary sequence
to targets are immobilized on a surface and the presence of target forms hybrids that are transduced
via electrochemical5253 or optical strategies54 Nano-surfaces can be integrated into this detection
strategy with oligonucleotides immobilized on the surface of nanoparticles allowing for
transduction via near-field phenomenon
13 Quantum dots
Nanomaterials based on gold and semiconductor composites have had a significant impact
across many different research fields including the chemical physical and biological sciences
Interest in nanoparticles has been driven due to the unique fundamental properties of these
materials as they approach and occupy size regions between bulk material and isolated atoms
Luminescent semiconductor-based quantum dots (QDs) in particular have attracted attention due
to their unique electro-optical properties arising from small size scales (typically ranging from
2 ndash 10 nm and consisting of 102 ndash 104 atoms per crystal) The key factors of interest for these
particles are material composition and size with a combination of the two giving rise to control of
physical properties such as the spectral profile and photon band gap energies55ndash59
There are many strategies for preparing and tuning the electro-optical properties of QDs
but some of the most studied from a synthetic perspective are based on binary composites of
elements from groups II-VI (like CdSe CdS or CdTe) and III-V (like InAs)55ndash58 For binary
composites luminescent properties can be controlled by choice of materials (selecting specific
regions of the UV-vis spectrum) and by control of size whereby smaller crystals are blue-shifted
and larger crystals are red-shifted5759 QDs used in most biological investigations are constructed
in a coreshell manner where the core is composed on a composite of materials previously
mentioned and the shell is composed of an inert coating (ie ZnS see Figure 2A) The QD shell
protects the nanoparticle from environmental degradation forming a protective layer and provides
a larger potential energy barrier for confining the exciton The shell material also provides a
synthetic strategy for controlling the core size and the type of shell allows for designing a class of
ligands for functionalizing the nanoparticle5556
9
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of
ZnS B Quantum dots of different colors are presented with their corresponding core size
image of solution and photoluminescence spectra and color C Diagram representing the
quantum confinement and the change in band gap energy as material size decreases below
the Bohr-exciton radius Here CB and VB represent the conduction and valence band
respectively and Eg represent the band gap energies Image adapted with permission
Copyright 2011 American Chemical Society60
The resulting particles have been incorporated into biological systems using surface ligands
with chemistry that allows the crystals to be stable in aqueous and physiological buffers6162
Further functionalization of these ligands has also allowed for the integration of biomolecules like
nucleic acids63 and proteins64 and polymers like polyethylene glycol (PEG) allowing applications
that extend from biological imaging65 to diagnostic device development and commercial
technologies566667 Optically quantum dots (QD) have broad absorption wavelengths (from the
UV into the visible) narrow and symmetrical emission photoluminescence (PL) profiles (25 nm
of full width at half maximum) high quantum yields and photochemical stability59 These
spectral properties in addition to the large surface area of QDs make them favourable donors for
RET processes
10
131 Quantum Confinement and The Particle in a Box
A brief overview of the quantum mechanics related to QDs will be discussed before
detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids
As the semiconducting material that composes QDs transitions from the bulk scale to the nano-
scale the valence and conductance bands of the semiconductor material split into discrete
energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the
composite of materials however for nanomaterials the band gap can also be tuned by modulating
the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the
dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like
CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an
electron-hole pair When an electron is excited by a photon of greater energy than the band gap
(the probability increases at higher energies yielding broad absorption spectra) the separation of
the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used
to describe this phenomenon is called quantum confinement and the model that best describes it is
the particle in a box575960
In this model a particle is said to be confined in a symmetrical box (of diameter a) where
the center of the box is denoted as = 0 and the edges of the box are denoted as = (
( Here
the potential energy inside the box +( le le
(- is said to be zero and the potential energy outside
the box + le ( ge
(- is said to be infinite The resulting probability of finding a particle outside
the confines of the box is zero 0 = 0 + le ( ge
(-1 and the discrete energy
eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of
interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material
The surface of the material represents the impenetrable barrier (potential energy is infinity)
restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869
As size of the QDs decreases the energy required for excitation increases because the
exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral
properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a
RET based system because the surface area of QDs allows for loading of multiple biomolecules
Thus multiple pathways of RET can take place that can collectively improve energy transfer
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
v
212 Instrumentation 24
22 Results and Discussion 25
221 FRET Pair Characterization (gQD ndash Cy3) 25
222 Oligonucleotide Hybridization in Solution 26
223 Oligonucleotide Hybridization in Paper Substrates 28
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by
Smartphone Imaging 32
225 Analytical Figures of Merit 38
226 Selectivity for Mixtures of WT and MT Targets 40
227 Paper-based Assay Response for Complex Sample Matrices 42
228 Blind Assay for Detection and Quantification of CFTR Target Mixes 43
Chapter 3 45
Conclusion and Future Work 45
31 Future Directions 46
References 47
vi
List of Tables
Table 1 Summary of FDA approved Nucleic Acid Based Tests for Diagnosis of CF 2
Table 2 Oligonucleotide Sequences used in Hybridization Assays 20
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids 34
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids 34
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids 34
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids 34
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids 36
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids 36
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids 36
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids 37
Table 11 Analytical Performance Direct and Sandwich Bioassays 40
Table 12 Blind Assay for Direct and Sandwich Assays 44
vii
List of Figures
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and Crick
Arrows on the ribbons represent the directionality bias for the single strands and dimensions for
the polymer are presented with one turn of the helix every 34 nm the distance between base pairs
every 034 nm and the distance between the phosphate backbone and the central axis every 1 nm
B shows the hydrogen bonding taking place between complementary pairs of nucleobases as
proposed by Chargaff with adenine (A) having two hydrogen bonds with thymine (T) and guanine
(G) having three hydrogen bonds with cytosine (C) Image was adapted with permission
Copyright Nature Education 201331 5
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of ZnS
B Quantum dots of different colors are presented with their corresponding core size image of
solution and photoluminescence spectra and color C Diagram representing the quantum
confinement and the change in band gap energy as material size decreases below the Bohr-exciton
radius Here CB and VB represent the conduction and valence band respectively and Eg represent
the band gap energies Image adapted with permission Copyright 2011 American Chemical
Society60 9
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of colloidally
stable and spherical QD (green) with multiple FRET acceptors (yellow) (b) Change in FRET
efficiency based on changes in the distance between donor and acceptor (c) QD (green)
immobilized on a surface can interact with multiple FRET acceptors by interacting with adjacent
acceptors Image acquired with permission from Algar et al70 Copyright Elsevier 2010 12
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in blue)
are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3 on
the proximal end and upon hybridization is brought to proximity with gQDs allowing for FRET
to take place (B) In sandwich assay format the probe strand hybridizes with the target strand (seen
in red) such that there is an overhang on the distal end Reporter strand (seen in green) hybridizes
with the overhang region of the target strand bringing to proximity the Cy3 label on the proximal
end of the reporter 14
viii
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society of
Chemistry 2016 16
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A) Reaction
zones consisted of chemically modified paper that were conjugated with gQD-oligonucleotide
probes Zones contained WT and MT controls and test zones where unknown samples were
spotted and imaged Detection was based on the principle of RET with gQDs used as donors and
Cy3 labels on oligonucleotide strands as acceptors (B) Imaging used a smartphone camera with
data processing by ImageJ to split the image to RGB color channels 18
Figure 7 Image of buffer solution leakage from hydrophilic paper zones 23
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines and
emission is shown as solid lines 26
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by fluorescence
spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii) CFTR single
DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT target strands The
concentration-response curves for the different gQD-probe conjugates are shown A WT Cy3
labelled target strands are seen in blue and MT Cy3 labelled target strands are seen in orange
Normalized PL spectra for the calibration curves are shown for B) CFTR WT Cy3 labelled target
strands and C) CFTR MT Cy3 labelled target strands ( indicates increasing target concentration)
27
Figure 10 Representations of the two different direct assay formats investigated in solution phase
gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA MT probe
and were mixed with complementary CFTR WT Cy3 target strands and CFTR MT Cy3 target
strands Hybridization resulted in proximity of gQDs and Cy3 which resulted in FRET 28
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of Cy3
labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol (vii) 75
ix
pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of spots that
may not be visible otherwise 29
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers to
WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash MT target
(D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and Tm) were calculated
using the nearest neighbor method3839 30
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash MT target
(D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and Tm) were calculated
using the nearest neighbor method3839 32
Figure 14 Determination of optimal wash conditions for direct and sandwich assay considered
RG Ratios with variation of formamide concentration for wash times of 0 5 10 15 and 20 min
The optimal wash conditions for direct assay was found to be BB+10F for 5 minutes for (A)
gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal wash conditions for
sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-WT probe sequence and
BB+5F for 20 minutes for (D) gQD-MT probe sequence 38
Figure 15 Concentration-response curves showing the RG ratiometric response of the direct and
sandwich assay formats (Ai) gQD-WT probe conjugates were used for determination of Cy3
labelled WT targets and (Bi) gQD-MT probe conjugates were used for determination of Cy3
labelled MT targets (Ci) gQD-WT probe conjugates were used for determination of unlabelled
WT targets with Cy3 labelled reporters and (Di) gQD-MT probe conjugates were used for
determination of unlabelled MT targets with Cy3 labelled reporters The RG ratiometric response
of the direct assay at the low pmol concentration range was also determined (Aii) gQD-WT probe
conjugates and (Bii) gQD-MT probe conjugates The sandwich assay format (Cii) gQD-WT probe
conjugates and (Dii) gQD-MT probe conjugates Note that the scale for (A) and (B) is logarithmic
Each error bar represents one standard deviation for n=4 replicates 39
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes and
(Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined using
background corrected RG ratio plots for hybridization of gQD-probe conjugates with Cy3 labelled
x
targets (for direct assay A and B) and gQD-probe conjugates with unlabeled targets and Cy3
labelled reporter sequences (for sandwich assay C and D) Response of the hybridization assay
was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-wash (Bi and Di) MT
probe conjugates Post-wash assays yielded signal response shown in Aii and Cii for WT probe
conjugates and in Bii and Dii for MT probe conjugates Error bars represent one standard deviation
for n = 4 replicates 41
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates and
(B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to direct assay
and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was collected for (C)
gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars represent one standard
deviation for n = 4 replicates 42
1
Chapter 1
Introduction
11 Cystic Fibrosis and Genes Associated with Cystic Fibrosis Transmembrane Protein
Cystic fibrosis (CF) is a multi-system fatal autosomal recessive disorder that is
characterized by viscous secretions in the lungs of patients due to mutations in cystic fibrosis
transmembrane conductance regulator protein (CFTR) CF affects 1 in 3000 births with ~70000
people affected worldwide1ndash5 Over 1500 mutations for the CFTR protein have been found but few
are common and fewer result in the disease Of the few mutations responsible for the disease state
the deletion of phenylalanine at the 508 position (∆F508) is responsible for over two-thirds of the
cases while all other mutations account for no more than 5 of the cases individually256
Development of sensing technology for early detection of ∆F508 would serve to enable improved
screening by clinicians to identify the predominant gene carriers The strategies for diagnosing CF
are based on newborn screening (NBS) programs that work via screening for serum markers
including the immunoreactive trypsinogen (IRT) assay7ndash9 This assay is typically followed by
diagnosis of the genetic basis of disease including detection of ∆F508 and related mutations based
on determining the presence of specific oligonucleotide sequences Finally a sweat chloride test
is performed to diagnose patients with CF All of these techniques require skilled technicians to
process samples perform and analyse tests via resource-intensive technologies10 The aim of this
work is to contribute to the development of a low cost easy to use and portable method for sensing
CFTR ∆F508 gene mutations beginning with a focus on a suitable transduction strategy
111 Nucleic Acids based Detection of Genes Associated with CF and ∆F508 mutation of CFTR Gene
There are multiple strategies for transducing the presence of genes associated with CF and
some of the technologies that have been approved by the United Stated Food and Drug
Administration (FDA) for use as in-vitro medical devices are presented in Table 1 (accessed Feb
20th 2018)11
2
Table 1 Summary of FDA approved Nucleic Acid Based Tests for Diagnosis of CF
Manufacturer Trade Name Detection Strategy
Illumina Inc Illumina MiSeqDx Cystic
Fibrosis Clinical Sequencing
Assay
Next-gen sequencing by
synthesis
Illumina MiSeqDx Cystic
Fibrosis 139-Variant Assay
Luminex Molecular
Diagnostics Inc
xTAG Cystic Fibrosis 60 kit v2 Microbead-dye barcode
coupled microarray analysis xTAG Cystic Fibrosis 39 kit v2
Osmetech Molecular
Diagnostics
eSensor CF Genotyping Test Sandwich hybridization assay
with ferrocene tag for cyclic
voltammetry analysis
Nanosphere Inc Verigene CFTR and Verigene
CFTR PolyT Nucleic Acid Tests
Genomic amplification
followed by sandwich assay
with probes and gold
nanoparticle reporters for
analysis
Third Wave Technology Inc InPlex CF Molecular Test PCR coupled with FRET based
microwell plate
Celera Diagnostics Cystic Fibrosis Genotyping
Assay
PCR coupled with capillary
electrophoresis and
oligonucleotide ligation assay
Typically these technologies require the use of specialized facilities and dedicated
technicians for analysis of patient samples and confirmation of CF may take up to a few weeks79
The resources and time required to diagnose patients may be reduced through the development of
point-of-care (POC) devices In particular the use of paper-based test strips with smartphone
detection for on-site rapid screening of disease markers would serve to alleviate the burden placed
on the health care system by more expensive techniques12
At the core of POC technology is the transduction strategy and much effort has gone into
developing optical13 and electrochemical methods14 for generating and measuring signal Yet the
application of this technology has not been investigated for selective sensing of similar nucleic
acid sequences that are often found to be associated with the genetic basis of disease Thus to
further discuss the challenges in this field it is important to address some of the background
technology that has been developed for POC sensors In particular this chapter will discuss nucleic
acid detection and the thermodynamics associated with hybridization interactions the use of
3
formamide as a chaotrope for controlling hybridization the use of nanomaterials like quantum dots
as integrated components in the bioassays for fluorescence resonance energy transfer-based
sensing strategies and the application of paper as a platform and substrate for sensing
12 Nucleic Acids and Oligonucleotide Detection
Deoxyribonucleic acid (DNA) is a class of biopolymers that stores hereditary information
and ribonucleic acid (RNA) functions as the set of instructions for synthesis of proteins15 The two-
step process by which the DNA nucleobase sequence is transcribed for production of RNA and
subsequently RNA is used as a template for translation to produce proteins is referred to as the
central dogma of molecular biology16 Proteins carry out the function that is encoded in the genetic
regions of DNA by interacting with other molecules and biopolymers present within and on the
surface of cells The specific interactions that govern the proteinsrsquo function are due to the three-
dimensional structure of the amino acid sequence that composes proteins17 The order of amino
acids which composes proteins is based on the nucleobase sequence of transcribing RNA (and
therefore DNA) Thus hereditary information stored as the base sequence of DNA can govern the
sequence of amino acids and therefore the structure and function of proteins1617 There are
numerous types of diseases that have arisen due to nucleobase-pair mutations in the sequence of
gene coding regions of DNA18 Mutations of DNA bases influence the amino acid sequence that
compose proteins and a three-base pair deletion like the one found for phenylalanine at codon 508
significantly alters the function of the protein associated with the CFTR gene Other types of
genetic diseases also arise due to mutations of the base pair sequence associated with DNA and
strategies for detection of nucleic acid mutations offer a method to detect the presence of a disease
state
To determine the genetic basis of disease for guiding clinical treatment diagnostic
technology for sensing nucleic acids must be further developed The main goal of clinical
diagnostic technology is to determine the molecular basis of disease for guiding patient therapy
because knowledge obtained from diagnostics are paramount for programing treatment strategies
Clinical diagnostics using a POC strategy offers improved opportunity for wide-scale screening
due to the advantages of low cost ease of manufacturing ease of transport ease of use and ease
of disposal19 One approach to the detection of genetic materials (deoxyribonucleic acid) is based
4
on hybridization and this process requires consideration of the chemical composition structure
and thermodynamics associated with hybridization
121 Structure and Composition of DNA Hybridization
Elucidation of DNArsquos structure and function has a long-storied history that has impacted
many fields of research including chemistry biology and medicine Much of the early work
related to DNA was focused on the structure of DNA with scientists focusing on the key details
related to the chemical composition of the monomers and the structural format of the polymeric
structure of DNA20ndash24 The key findings of the structure of DNA are summarized as follows
1 The structure for the DNA salt is composed of two helical polymer chains that are
coiled around one another and around a shared axis (see Figure 1A) The outside of the
chains is composed of phosphate-sugars groups and the chains are linked together on
the inside via hydrogen bonds between the nucleotide bases
2 The chains are anti-parallel in conformation with ie the 5rsquo end of one chain is bound
via the nucleobases to the 3rsquo end of the other chain
3 Both chains follow a right-handed helix (one type of DNA known as Z-DNA follows
a left-handed helix but this was discovered later)25 and base residues are present on the
chains every 34 Å with structural repeats every ten residues The distance from the
central shared axis to the phosphorous atom is 10 Å
4 The four bases composing DNA bond to a specific pair (see Figure 1B) ie adenine
(purine) binds with thymine (pyrimidine) and guanine (purine) binds with cytosine
(pyrimidine) The relationship of molar equivalency between pairs of bases ie A ndash T
and G ndash C was determined earlier by Chargaff in 195026
Details related to the structure and composition of DNA has formed the basis of our
understanding of the role of DNA in molecular and cell biology Through the structure of DNA
the mechanism for DNA replication27 transcription28 and translation29 for protein synthesis was
elucidated The confirmation of DNA as the storage for hereditary information paved the way for
initiatives like the Human Genome Project and insights from this undertaking have fueled research
regarding the genetic basis of disease30
5
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and
Crick Arrows on the ribbons represent the directionality bias for the single strands and
dimensions for the polymer are presented with one turn of the helix every 34 nm the
distance between base pairs every 034 nm and the distance between the phosphate
backbone and the central axis every 1 nm B shows the hydrogen bonding taking place
between complementary pairs of nucleobases as proposed by Chargaff with adenine (A)
having two hydrogen bonds with thymine (T) and guanine (G) having three hydrogen bonds
with cytosine (C) Image was adapted with permission Copyright Nature Education 201331
122 Thermodynamics of DNA Hybridization
Design and development of DNA-based technologies have been guided by the
thermodynamic modelling of hybridization Techniques like PCR3233 and isothermal
amplification34 rely on accurate control over the annealing of primers and DNA sensors often uses
temperature and chaotropic agents for achieving selectivity35 One of the useful tools for modelling
hybridization and mismatch-based interactions is the nearest neighbor method (NN)36 To explore
the strategy between the NN method and hybridization of DNA it is useful to understand some
details related to predicting the melting temperature (Tm)
First the system of interest will be defined at the equilibrium of dsDNA and ssDNA at the
point where both populations are equal ie half the strands of DNA are in the double helix state
and the other half are single-stranded and are often in various conformations Tm is the temperature
6
at which this equilibrium is found Next the equilibrium constant Keq is defined as being a ratio
of the concentration of dsDNA and ssDNA (as stated in Equation 1) A second expression can be
derived from the Vanrsquot Hoff equation (as stated in Equation 2) where ∆Ho and ∆So are the standard
enthalpy and entropy of hybridization and lnCT is the natural logarithm of the total strand
concentration This second equation can be used to calculate the thermodynamic parameters
related to Tm with the same being true vice versa37
Equation 1 = [][]
Equation 2 = ∆∆
With this foundation investigation into the NN method for modelling can be undertaken
The thermodynamics associated with a base pair are related to some degree with neighboring base
pairs Free energy values and other related parameters have been determined experimentally for
model oligonucleotide sequences This information is then used in conjunction with the nearest
neighbor algorithm (as presented in Equation 3) for obtaining the Tm for the strand of interest
Here base pair doublets are considered for sequence stability with ten unique combinations of
doublets (5rsquo-3rsquo) CG GC AT AA (also = TT) AG (also = CT) AC (also = GT) GA (also = TC)
GG (also = CC) TG (also = CA)38
Equation 3 ∆ = ∆ + ∆ + sum ∆
Equation 4 ∆ = ∆ minus ∆
In Equation 3 the ∆Gi(total) refers to the free energy of the strand of interest ∆G(init)
refers to the free energy of the strand of initiating base pair ∆G(sym) refers to the free energy of
symmetry Gj refers to the free energy associated with one of ten nearest neighbor stacking
interactions and nij is the appearance rate of the stacking interaction of interest Thermodynamic
parameters are also present for entropy and enthalpy allowing for the calculation of the Tm using
Equation 43638 Higher Tm values indicate greater stability than lower Tm values39ndash42 The NN
method can also be used along with a database of mismatch energetics to determine the
thermodynamic parameters related to those sequences
7
Tm values when used in conjunction with the free energies provide a theoretical basis for
designing probe ndash capture strand interactions This understanding can be useful when designing
wash conditions that control stringency for oligonucleotides composed of sequences with high
similarity Stringency control can be achieved using higher temperature (because increasing
temperature results in de-annealing of sequences and has greater effect on hybrids with partial
complementarity)43 by controlling the ionic strength of hybridization44 and via chaotropic agents
such as formamide45 and urea46 Although all strategies are valid for reducing false positive signals
(that arise from partially complementary strands of oligonucleotides) the use of washes containing
chaotropic agents may be more applicable for the POC given that temperature control requires a
temperature module
Chaotropic agents like formamide lower the melting temperature of duplex DNA by
engaging with the hydrogen bond network of DNA The degree by which temperature is lowered
depends on the GC content the conformations of single and duplex forms and the hydration state
of the strand (but typically can range 24 ndash 29 degC per mole of formamide)45 Chemically
formamide strongly associates with DNA is capable of four hydrogen bonds (same as water) and
is a stronger hydrogen bond acceptor than water Formamide ndash water bonds have been reported to
be 20 stronger than water ndash water bonds and it is accepted that formamide engages the hydration
network around DNA4547 Thus using formamide in washes for DNA hybridization can lower the
melting temperature favoring fully complementary hybrids over partially complementary hybrids
123 Notes and Considerations for POC Application
Developing a DNA screening device for the POC application requires consideration of the
many challenges faced by clinicians When screening genetic samples from blood it is important
to note that samples are often complex with proteins and other type of biomolecules (in addition
to cellular debris) and these materials may occlude the signal generated from target detection48
Another challenge to note for nucleic acid-based diagnostics is the low amount of target present in
clinical samples For example one milliliter of human blood contains approximately 107
leukocytes corresponding to femtomolar quantities (fM or attomoles 10-18 moles) of target nucleic
acid Thus detection strategies requiring hybridization-based assay require enzymatic
amplification of the target materials or improved analytical figures of merit for application in
POC49 Presently there are many different strategies for enzymatic amplification of nucleic acids
8
including polymerase chain reaction32 loop-mediated isothermal amplification34 helicase-
dependent isothermal amplification50 and recombinase polymerase amplification51 Post
amplification targets are often detected using hybridization-based assays using Watson-Crick base
pairing for detection of targets of interest Typically capture probes of complementary sequence
to targets are immobilized on a surface and the presence of target forms hybrids that are transduced
via electrochemical5253 or optical strategies54 Nano-surfaces can be integrated into this detection
strategy with oligonucleotides immobilized on the surface of nanoparticles allowing for
transduction via near-field phenomenon
13 Quantum dots
Nanomaterials based on gold and semiconductor composites have had a significant impact
across many different research fields including the chemical physical and biological sciences
Interest in nanoparticles has been driven due to the unique fundamental properties of these
materials as they approach and occupy size regions between bulk material and isolated atoms
Luminescent semiconductor-based quantum dots (QDs) in particular have attracted attention due
to their unique electro-optical properties arising from small size scales (typically ranging from
2 ndash 10 nm and consisting of 102 ndash 104 atoms per crystal) The key factors of interest for these
particles are material composition and size with a combination of the two giving rise to control of
physical properties such as the spectral profile and photon band gap energies55ndash59
There are many strategies for preparing and tuning the electro-optical properties of QDs
but some of the most studied from a synthetic perspective are based on binary composites of
elements from groups II-VI (like CdSe CdS or CdTe) and III-V (like InAs)55ndash58 For binary
composites luminescent properties can be controlled by choice of materials (selecting specific
regions of the UV-vis spectrum) and by control of size whereby smaller crystals are blue-shifted
and larger crystals are red-shifted5759 QDs used in most biological investigations are constructed
in a coreshell manner where the core is composed on a composite of materials previously
mentioned and the shell is composed of an inert coating (ie ZnS see Figure 2A) The QD shell
protects the nanoparticle from environmental degradation forming a protective layer and provides
a larger potential energy barrier for confining the exciton The shell material also provides a
synthetic strategy for controlling the core size and the type of shell allows for designing a class of
ligands for functionalizing the nanoparticle5556
9
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of
ZnS B Quantum dots of different colors are presented with their corresponding core size
image of solution and photoluminescence spectra and color C Diagram representing the
quantum confinement and the change in band gap energy as material size decreases below
the Bohr-exciton radius Here CB and VB represent the conduction and valence band
respectively and Eg represent the band gap energies Image adapted with permission
Copyright 2011 American Chemical Society60
The resulting particles have been incorporated into biological systems using surface ligands
with chemistry that allows the crystals to be stable in aqueous and physiological buffers6162
Further functionalization of these ligands has also allowed for the integration of biomolecules like
nucleic acids63 and proteins64 and polymers like polyethylene glycol (PEG) allowing applications
that extend from biological imaging65 to diagnostic device development and commercial
technologies566667 Optically quantum dots (QD) have broad absorption wavelengths (from the
UV into the visible) narrow and symmetrical emission photoluminescence (PL) profiles (25 nm
of full width at half maximum) high quantum yields and photochemical stability59 These
spectral properties in addition to the large surface area of QDs make them favourable donors for
RET processes
10
131 Quantum Confinement and The Particle in a Box
A brief overview of the quantum mechanics related to QDs will be discussed before
detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids
As the semiconducting material that composes QDs transitions from the bulk scale to the nano-
scale the valence and conductance bands of the semiconductor material split into discrete
energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the
composite of materials however for nanomaterials the band gap can also be tuned by modulating
the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the
dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like
CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an
electron-hole pair When an electron is excited by a photon of greater energy than the band gap
(the probability increases at higher energies yielding broad absorption spectra) the separation of
the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used
to describe this phenomenon is called quantum confinement and the model that best describes it is
the particle in a box575960
In this model a particle is said to be confined in a symmetrical box (of diameter a) where
the center of the box is denoted as = 0 and the edges of the box are denoted as = (
( Here
the potential energy inside the box +( le le
(- is said to be zero and the potential energy outside
the box + le ( ge
(- is said to be infinite The resulting probability of finding a particle outside
the confines of the box is zero 0 = 0 + le ( ge
(-1 and the discrete energy
eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of
interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material
The surface of the material represents the impenetrable barrier (potential energy is infinity)
restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869
As size of the QDs decreases the energy required for excitation increases because the
exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral
properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a
RET based system because the surface area of QDs allows for loading of multiple biomolecules
Thus multiple pathways of RET can take place that can collectively improve energy transfer
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
vi
List of Tables
Table 1 Summary of FDA approved Nucleic Acid Based Tests for Diagnosis of CF 2
Table 2 Oligonucleotide Sequences used in Hybridization Assays 20
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids 34
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids 34
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids 34
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids 34
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids 36
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids 36
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids 36
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids 37
Table 11 Analytical Performance Direct and Sandwich Bioassays 40
Table 12 Blind Assay for Direct and Sandwich Assays 44
vii
List of Figures
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and Crick
Arrows on the ribbons represent the directionality bias for the single strands and dimensions for
the polymer are presented with one turn of the helix every 34 nm the distance between base pairs
every 034 nm and the distance between the phosphate backbone and the central axis every 1 nm
B shows the hydrogen bonding taking place between complementary pairs of nucleobases as
proposed by Chargaff with adenine (A) having two hydrogen bonds with thymine (T) and guanine
(G) having three hydrogen bonds with cytosine (C) Image was adapted with permission
Copyright Nature Education 201331 5
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of ZnS
B Quantum dots of different colors are presented with their corresponding core size image of
solution and photoluminescence spectra and color C Diagram representing the quantum
confinement and the change in band gap energy as material size decreases below the Bohr-exciton
radius Here CB and VB represent the conduction and valence band respectively and Eg represent
the band gap energies Image adapted with permission Copyright 2011 American Chemical
Society60 9
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of colloidally
stable and spherical QD (green) with multiple FRET acceptors (yellow) (b) Change in FRET
efficiency based on changes in the distance between donor and acceptor (c) QD (green)
immobilized on a surface can interact with multiple FRET acceptors by interacting with adjacent
acceptors Image acquired with permission from Algar et al70 Copyright Elsevier 2010 12
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in blue)
are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3 on
the proximal end and upon hybridization is brought to proximity with gQDs allowing for FRET
to take place (B) In sandwich assay format the probe strand hybridizes with the target strand (seen
in red) such that there is an overhang on the distal end Reporter strand (seen in green) hybridizes
with the overhang region of the target strand bringing to proximity the Cy3 label on the proximal
end of the reporter 14
viii
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society of
Chemistry 2016 16
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A) Reaction
zones consisted of chemically modified paper that were conjugated with gQD-oligonucleotide
probes Zones contained WT and MT controls and test zones where unknown samples were
spotted and imaged Detection was based on the principle of RET with gQDs used as donors and
Cy3 labels on oligonucleotide strands as acceptors (B) Imaging used a smartphone camera with
data processing by ImageJ to split the image to RGB color channels 18
Figure 7 Image of buffer solution leakage from hydrophilic paper zones 23
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines and
emission is shown as solid lines 26
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by fluorescence
spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii) CFTR single
DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT target strands The
concentration-response curves for the different gQD-probe conjugates are shown A WT Cy3
labelled target strands are seen in blue and MT Cy3 labelled target strands are seen in orange
Normalized PL spectra for the calibration curves are shown for B) CFTR WT Cy3 labelled target
strands and C) CFTR MT Cy3 labelled target strands ( indicates increasing target concentration)
27
Figure 10 Representations of the two different direct assay formats investigated in solution phase
gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA MT probe
and were mixed with complementary CFTR WT Cy3 target strands and CFTR MT Cy3 target
strands Hybridization resulted in proximity of gQDs and Cy3 which resulted in FRET 28
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of Cy3
labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol (vii) 75
ix
pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of spots that
may not be visible otherwise 29
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers to
WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash MT target
(D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and Tm) were calculated
using the nearest neighbor method3839 30
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash MT target
(D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and Tm) were calculated
using the nearest neighbor method3839 32
Figure 14 Determination of optimal wash conditions for direct and sandwich assay considered
RG Ratios with variation of formamide concentration for wash times of 0 5 10 15 and 20 min
The optimal wash conditions for direct assay was found to be BB+10F for 5 minutes for (A)
gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal wash conditions for
sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-WT probe sequence and
BB+5F for 20 minutes for (D) gQD-MT probe sequence 38
Figure 15 Concentration-response curves showing the RG ratiometric response of the direct and
sandwich assay formats (Ai) gQD-WT probe conjugates were used for determination of Cy3
labelled WT targets and (Bi) gQD-MT probe conjugates were used for determination of Cy3
labelled MT targets (Ci) gQD-WT probe conjugates were used for determination of unlabelled
WT targets with Cy3 labelled reporters and (Di) gQD-MT probe conjugates were used for
determination of unlabelled MT targets with Cy3 labelled reporters The RG ratiometric response
of the direct assay at the low pmol concentration range was also determined (Aii) gQD-WT probe
conjugates and (Bii) gQD-MT probe conjugates The sandwich assay format (Cii) gQD-WT probe
conjugates and (Dii) gQD-MT probe conjugates Note that the scale for (A) and (B) is logarithmic
Each error bar represents one standard deviation for n=4 replicates 39
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes and
(Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined using
background corrected RG ratio plots for hybridization of gQD-probe conjugates with Cy3 labelled
x
targets (for direct assay A and B) and gQD-probe conjugates with unlabeled targets and Cy3
labelled reporter sequences (for sandwich assay C and D) Response of the hybridization assay
was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-wash (Bi and Di) MT
probe conjugates Post-wash assays yielded signal response shown in Aii and Cii for WT probe
conjugates and in Bii and Dii for MT probe conjugates Error bars represent one standard deviation
for n = 4 replicates 41
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates and
(B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to direct assay
and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was collected for (C)
gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars represent one standard
deviation for n = 4 replicates 42
1
Chapter 1
Introduction
11 Cystic Fibrosis and Genes Associated with Cystic Fibrosis Transmembrane Protein
Cystic fibrosis (CF) is a multi-system fatal autosomal recessive disorder that is
characterized by viscous secretions in the lungs of patients due to mutations in cystic fibrosis
transmembrane conductance regulator protein (CFTR) CF affects 1 in 3000 births with ~70000
people affected worldwide1ndash5 Over 1500 mutations for the CFTR protein have been found but few
are common and fewer result in the disease Of the few mutations responsible for the disease state
the deletion of phenylalanine at the 508 position (∆F508) is responsible for over two-thirds of the
cases while all other mutations account for no more than 5 of the cases individually256
Development of sensing technology for early detection of ∆F508 would serve to enable improved
screening by clinicians to identify the predominant gene carriers The strategies for diagnosing CF
are based on newborn screening (NBS) programs that work via screening for serum markers
including the immunoreactive trypsinogen (IRT) assay7ndash9 This assay is typically followed by
diagnosis of the genetic basis of disease including detection of ∆F508 and related mutations based
on determining the presence of specific oligonucleotide sequences Finally a sweat chloride test
is performed to diagnose patients with CF All of these techniques require skilled technicians to
process samples perform and analyse tests via resource-intensive technologies10 The aim of this
work is to contribute to the development of a low cost easy to use and portable method for sensing
CFTR ∆F508 gene mutations beginning with a focus on a suitable transduction strategy
111 Nucleic Acids based Detection of Genes Associated with CF and ∆F508 mutation of CFTR Gene
There are multiple strategies for transducing the presence of genes associated with CF and
some of the technologies that have been approved by the United Stated Food and Drug
Administration (FDA) for use as in-vitro medical devices are presented in Table 1 (accessed Feb
20th 2018)11
2
Table 1 Summary of FDA approved Nucleic Acid Based Tests for Diagnosis of CF
Manufacturer Trade Name Detection Strategy
Illumina Inc Illumina MiSeqDx Cystic
Fibrosis Clinical Sequencing
Assay
Next-gen sequencing by
synthesis
Illumina MiSeqDx Cystic
Fibrosis 139-Variant Assay
Luminex Molecular
Diagnostics Inc
xTAG Cystic Fibrosis 60 kit v2 Microbead-dye barcode
coupled microarray analysis xTAG Cystic Fibrosis 39 kit v2
Osmetech Molecular
Diagnostics
eSensor CF Genotyping Test Sandwich hybridization assay
with ferrocene tag for cyclic
voltammetry analysis
Nanosphere Inc Verigene CFTR and Verigene
CFTR PolyT Nucleic Acid Tests
Genomic amplification
followed by sandwich assay
with probes and gold
nanoparticle reporters for
analysis
Third Wave Technology Inc InPlex CF Molecular Test PCR coupled with FRET based
microwell plate
Celera Diagnostics Cystic Fibrosis Genotyping
Assay
PCR coupled with capillary
electrophoresis and
oligonucleotide ligation assay
Typically these technologies require the use of specialized facilities and dedicated
technicians for analysis of patient samples and confirmation of CF may take up to a few weeks79
The resources and time required to diagnose patients may be reduced through the development of
point-of-care (POC) devices In particular the use of paper-based test strips with smartphone
detection for on-site rapid screening of disease markers would serve to alleviate the burden placed
on the health care system by more expensive techniques12
At the core of POC technology is the transduction strategy and much effort has gone into
developing optical13 and electrochemical methods14 for generating and measuring signal Yet the
application of this technology has not been investigated for selective sensing of similar nucleic
acid sequences that are often found to be associated with the genetic basis of disease Thus to
further discuss the challenges in this field it is important to address some of the background
technology that has been developed for POC sensors In particular this chapter will discuss nucleic
acid detection and the thermodynamics associated with hybridization interactions the use of
3
formamide as a chaotrope for controlling hybridization the use of nanomaterials like quantum dots
as integrated components in the bioassays for fluorescence resonance energy transfer-based
sensing strategies and the application of paper as a platform and substrate for sensing
12 Nucleic Acids and Oligonucleotide Detection
Deoxyribonucleic acid (DNA) is a class of biopolymers that stores hereditary information
and ribonucleic acid (RNA) functions as the set of instructions for synthesis of proteins15 The two-
step process by which the DNA nucleobase sequence is transcribed for production of RNA and
subsequently RNA is used as a template for translation to produce proteins is referred to as the
central dogma of molecular biology16 Proteins carry out the function that is encoded in the genetic
regions of DNA by interacting with other molecules and biopolymers present within and on the
surface of cells The specific interactions that govern the proteinsrsquo function are due to the three-
dimensional structure of the amino acid sequence that composes proteins17 The order of amino
acids which composes proteins is based on the nucleobase sequence of transcribing RNA (and
therefore DNA) Thus hereditary information stored as the base sequence of DNA can govern the
sequence of amino acids and therefore the structure and function of proteins1617 There are
numerous types of diseases that have arisen due to nucleobase-pair mutations in the sequence of
gene coding regions of DNA18 Mutations of DNA bases influence the amino acid sequence that
compose proteins and a three-base pair deletion like the one found for phenylalanine at codon 508
significantly alters the function of the protein associated with the CFTR gene Other types of
genetic diseases also arise due to mutations of the base pair sequence associated with DNA and
strategies for detection of nucleic acid mutations offer a method to detect the presence of a disease
state
To determine the genetic basis of disease for guiding clinical treatment diagnostic
technology for sensing nucleic acids must be further developed The main goal of clinical
diagnostic technology is to determine the molecular basis of disease for guiding patient therapy
because knowledge obtained from diagnostics are paramount for programing treatment strategies
Clinical diagnostics using a POC strategy offers improved opportunity for wide-scale screening
due to the advantages of low cost ease of manufacturing ease of transport ease of use and ease
of disposal19 One approach to the detection of genetic materials (deoxyribonucleic acid) is based
4
on hybridization and this process requires consideration of the chemical composition structure
and thermodynamics associated with hybridization
121 Structure and Composition of DNA Hybridization
Elucidation of DNArsquos structure and function has a long-storied history that has impacted
many fields of research including chemistry biology and medicine Much of the early work
related to DNA was focused on the structure of DNA with scientists focusing on the key details
related to the chemical composition of the monomers and the structural format of the polymeric
structure of DNA20ndash24 The key findings of the structure of DNA are summarized as follows
1 The structure for the DNA salt is composed of two helical polymer chains that are
coiled around one another and around a shared axis (see Figure 1A) The outside of the
chains is composed of phosphate-sugars groups and the chains are linked together on
the inside via hydrogen bonds between the nucleotide bases
2 The chains are anti-parallel in conformation with ie the 5rsquo end of one chain is bound
via the nucleobases to the 3rsquo end of the other chain
3 Both chains follow a right-handed helix (one type of DNA known as Z-DNA follows
a left-handed helix but this was discovered later)25 and base residues are present on the
chains every 34 Å with structural repeats every ten residues The distance from the
central shared axis to the phosphorous atom is 10 Å
4 The four bases composing DNA bond to a specific pair (see Figure 1B) ie adenine
(purine) binds with thymine (pyrimidine) and guanine (purine) binds with cytosine
(pyrimidine) The relationship of molar equivalency between pairs of bases ie A ndash T
and G ndash C was determined earlier by Chargaff in 195026
Details related to the structure and composition of DNA has formed the basis of our
understanding of the role of DNA in molecular and cell biology Through the structure of DNA
the mechanism for DNA replication27 transcription28 and translation29 for protein synthesis was
elucidated The confirmation of DNA as the storage for hereditary information paved the way for
initiatives like the Human Genome Project and insights from this undertaking have fueled research
regarding the genetic basis of disease30
5
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and
Crick Arrows on the ribbons represent the directionality bias for the single strands and
dimensions for the polymer are presented with one turn of the helix every 34 nm the
distance between base pairs every 034 nm and the distance between the phosphate
backbone and the central axis every 1 nm B shows the hydrogen bonding taking place
between complementary pairs of nucleobases as proposed by Chargaff with adenine (A)
having two hydrogen bonds with thymine (T) and guanine (G) having three hydrogen bonds
with cytosine (C) Image was adapted with permission Copyright Nature Education 201331
122 Thermodynamics of DNA Hybridization
Design and development of DNA-based technologies have been guided by the
thermodynamic modelling of hybridization Techniques like PCR3233 and isothermal
amplification34 rely on accurate control over the annealing of primers and DNA sensors often uses
temperature and chaotropic agents for achieving selectivity35 One of the useful tools for modelling
hybridization and mismatch-based interactions is the nearest neighbor method (NN)36 To explore
the strategy between the NN method and hybridization of DNA it is useful to understand some
details related to predicting the melting temperature (Tm)
First the system of interest will be defined at the equilibrium of dsDNA and ssDNA at the
point where both populations are equal ie half the strands of DNA are in the double helix state
and the other half are single-stranded and are often in various conformations Tm is the temperature
6
at which this equilibrium is found Next the equilibrium constant Keq is defined as being a ratio
of the concentration of dsDNA and ssDNA (as stated in Equation 1) A second expression can be
derived from the Vanrsquot Hoff equation (as stated in Equation 2) where ∆Ho and ∆So are the standard
enthalpy and entropy of hybridization and lnCT is the natural logarithm of the total strand
concentration This second equation can be used to calculate the thermodynamic parameters
related to Tm with the same being true vice versa37
Equation 1 = [][]
Equation 2 = ∆∆
With this foundation investigation into the NN method for modelling can be undertaken
The thermodynamics associated with a base pair are related to some degree with neighboring base
pairs Free energy values and other related parameters have been determined experimentally for
model oligonucleotide sequences This information is then used in conjunction with the nearest
neighbor algorithm (as presented in Equation 3) for obtaining the Tm for the strand of interest
Here base pair doublets are considered for sequence stability with ten unique combinations of
doublets (5rsquo-3rsquo) CG GC AT AA (also = TT) AG (also = CT) AC (also = GT) GA (also = TC)
GG (also = CC) TG (also = CA)38
Equation 3 ∆ = ∆ + ∆ + sum ∆
Equation 4 ∆ = ∆ minus ∆
In Equation 3 the ∆Gi(total) refers to the free energy of the strand of interest ∆G(init)
refers to the free energy of the strand of initiating base pair ∆G(sym) refers to the free energy of
symmetry Gj refers to the free energy associated with one of ten nearest neighbor stacking
interactions and nij is the appearance rate of the stacking interaction of interest Thermodynamic
parameters are also present for entropy and enthalpy allowing for the calculation of the Tm using
Equation 43638 Higher Tm values indicate greater stability than lower Tm values39ndash42 The NN
method can also be used along with a database of mismatch energetics to determine the
thermodynamic parameters related to those sequences
7
Tm values when used in conjunction with the free energies provide a theoretical basis for
designing probe ndash capture strand interactions This understanding can be useful when designing
wash conditions that control stringency for oligonucleotides composed of sequences with high
similarity Stringency control can be achieved using higher temperature (because increasing
temperature results in de-annealing of sequences and has greater effect on hybrids with partial
complementarity)43 by controlling the ionic strength of hybridization44 and via chaotropic agents
such as formamide45 and urea46 Although all strategies are valid for reducing false positive signals
(that arise from partially complementary strands of oligonucleotides) the use of washes containing
chaotropic agents may be more applicable for the POC given that temperature control requires a
temperature module
Chaotropic agents like formamide lower the melting temperature of duplex DNA by
engaging with the hydrogen bond network of DNA The degree by which temperature is lowered
depends on the GC content the conformations of single and duplex forms and the hydration state
of the strand (but typically can range 24 ndash 29 degC per mole of formamide)45 Chemically
formamide strongly associates with DNA is capable of four hydrogen bonds (same as water) and
is a stronger hydrogen bond acceptor than water Formamide ndash water bonds have been reported to
be 20 stronger than water ndash water bonds and it is accepted that formamide engages the hydration
network around DNA4547 Thus using formamide in washes for DNA hybridization can lower the
melting temperature favoring fully complementary hybrids over partially complementary hybrids
123 Notes and Considerations for POC Application
Developing a DNA screening device for the POC application requires consideration of the
many challenges faced by clinicians When screening genetic samples from blood it is important
to note that samples are often complex with proteins and other type of biomolecules (in addition
to cellular debris) and these materials may occlude the signal generated from target detection48
Another challenge to note for nucleic acid-based diagnostics is the low amount of target present in
clinical samples For example one milliliter of human blood contains approximately 107
leukocytes corresponding to femtomolar quantities (fM or attomoles 10-18 moles) of target nucleic
acid Thus detection strategies requiring hybridization-based assay require enzymatic
amplification of the target materials or improved analytical figures of merit for application in
POC49 Presently there are many different strategies for enzymatic amplification of nucleic acids
8
including polymerase chain reaction32 loop-mediated isothermal amplification34 helicase-
dependent isothermal amplification50 and recombinase polymerase amplification51 Post
amplification targets are often detected using hybridization-based assays using Watson-Crick base
pairing for detection of targets of interest Typically capture probes of complementary sequence
to targets are immobilized on a surface and the presence of target forms hybrids that are transduced
via electrochemical5253 or optical strategies54 Nano-surfaces can be integrated into this detection
strategy with oligonucleotides immobilized on the surface of nanoparticles allowing for
transduction via near-field phenomenon
13 Quantum dots
Nanomaterials based on gold and semiconductor composites have had a significant impact
across many different research fields including the chemical physical and biological sciences
Interest in nanoparticles has been driven due to the unique fundamental properties of these
materials as they approach and occupy size regions between bulk material and isolated atoms
Luminescent semiconductor-based quantum dots (QDs) in particular have attracted attention due
to their unique electro-optical properties arising from small size scales (typically ranging from
2 ndash 10 nm and consisting of 102 ndash 104 atoms per crystal) The key factors of interest for these
particles are material composition and size with a combination of the two giving rise to control of
physical properties such as the spectral profile and photon band gap energies55ndash59
There are many strategies for preparing and tuning the electro-optical properties of QDs
but some of the most studied from a synthetic perspective are based on binary composites of
elements from groups II-VI (like CdSe CdS or CdTe) and III-V (like InAs)55ndash58 For binary
composites luminescent properties can be controlled by choice of materials (selecting specific
regions of the UV-vis spectrum) and by control of size whereby smaller crystals are blue-shifted
and larger crystals are red-shifted5759 QDs used in most biological investigations are constructed
in a coreshell manner where the core is composed on a composite of materials previously
mentioned and the shell is composed of an inert coating (ie ZnS see Figure 2A) The QD shell
protects the nanoparticle from environmental degradation forming a protective layer and provides
a larger potential energy barrier for confining the exciton The shell material also provides a
synthetic strategy for controlling the core size and the type of shell allows for designing a class of
ligands for functionalizing the nanoparticle5556
9
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of
ZnS B Quantum dots of different colors are presented with their corresponding core size
image of solution and photoluminescence spectra and color C Diagram representing the
quantum confinement and the change in band gap energy as material size decreases below
the Bohr-exciton radius Here CB and VB represent the conduction and valence band
respectively and Eg represent the band gap energies Image adapted with permission
Copyright 2011 American Chemical Society60
The resulting particles have been incorporated into biological systems using surface ligands
with chemistry that allows the crystals to be stable in aqueous and physiological buffers6162
Further functionalization of these ligands has also allowed for the integration of biomolecules like
nucleic acids63 and proteins64 and polymers like polyethylene glycol (PEG) allowing applications
that extend from biological imaging65 to diagnostic device development and commercial
technologies566667 Optically quantum dots (QD) have broad absorption wavelengths (from the
UV into the visible) narrow and symmetrical emission photoluminescence (PL) profiles (25 nm
of full width at half maximum) high quantum yields and photochemical stability59 These
spectral properties in addition to the large surface area of QDs make them favourable donors for
RET processes
10
131 Quantum Confinement and The Particle in a Box
A brief overview of the quantum mechanics related to QDs will be discussed before
detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids
As the semiconducting material that composes QDs transitions from the bulk scale to the nano-
scale the valence and conductance bands of the semiconductor material split into discrete
energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the
composite of materials however for nanomaterials the band gap can also be tuned by modulating
the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the
dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like
CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an
electron-hole pair When an electron is excited by a photon of greater energy than the band gap
(the probability increases at higher energies yielding broad absorption spectra) the separation of
the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used
to describe this phenomenon is called quantum confinement and the model that best describes it is
the particle in a box575960
In this model a particle is said to be confined in a symmetrical box (of diameter a) where
the center of the box is denoted as = 0 and the edges of the box are denoted as = (
( Here
the potential energy inside the box +( le le
(- is said to be zero and the potential energy outside
the box + le ( ge
(- is said to be infinite The resulting probability of finding a particle outside
the confines of the box is zero 0 = 0 + le ( ge
(-1 and the discrete energy
eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of
interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material
The surface of the material represents the impenetrable barrier (potential energy is infinity)
restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869
As size of the QDs decreases the energy required for excitation increases because the
exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral
properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a
RET based system because the surface area of QDs allows for loading of multiple biomolecules
Thus multiple pathways of RET can take place that can collectively improve energy transfer
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
vii
List of Figures
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and Crick
Arrows on the ribbons represent the directionality bias for the single strands and dimensions for
the polymer are presented with one turn of the helix every 34 nm the distance between base pairs
every 034 nm and the distance between the phosphate backbone and the central axis every 1 nm
B shows the hydrogen bonding taking place between complementary pairs of nucleobases as
proposed by Chargaff with adenine (A) having two hydrogen bonds with thymine (T) and guanine
(G) having three hydrogen bonds with cytosine (C) Image was adapted with permission
Copyright Nature Education 201331 5
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of ZnS
B Quantum dots of different colors are presented with their corresponding core size image of
solution and photoluminescence spectra and color C Diagram representing the quantum
confinement and the change in band gap energy as material size decreases below the Bohr-exciton
radius Here CB and VB represent the conduction and valence band respectively and Eg represent
the band gap energies Image adapted with permission Copyright 2011 American Chemical
Society60 9
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of colloidally
stable and spherical QD (green) with multiple FRET acceptors (yellow) (b) Change in FRET
efficiency based on changes in the distance between donor and acceptor (c) QD (green)
immobilized on a surface can interact with multiple FRET acceptors by interacting with adjacent
acceptors Image acquired with permission from Algar et al70 Copyright Elsevier 2010 12
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in blue)
are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3 on
the proximal end and upon hybridization is brought to proximity with gQDs allowing for FRET
to take place (B) In sandwich assay format the probe strand hybridizes with the target strand (seen
in red) such that there is an overhang on the distal end Reporter strand (seen in green) hybridizes
with the overhang region of the target strand bringing to proximity the Cy3 label on the proximal
end of the reporter 14
viii
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society of
Chemistry 2016 16
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A) Reaction
zones consisted of chemically modified paper that were conjugated with gQD-oligonucleotide
probes Zones contained WT and MT controls and test zones where unknown samples were
spotted and imaged Detection was based on the principle of RET with gQDs used as donors and
Cy3 labels on oligonucleotide strands as acceptors (B) Imaging used a smartphone camera with
data processing by ImageJ to split the image to RGB color channels 18
Figure 7 Image of buffer solution leakage from hydrophilic paper zones 23
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines and
emission is shown as solid lines 26
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by fluorescence
spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii) CFTR single
DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT target strands The
concentration-response curves for the different gQD-probe conjugates are shown A WT Cy3
labelled target strands are seen in blue and MT Cy3 labelled target strands are seen in orange
Normalized PL spectra for the calibration curves are shown for B) CFTR WT Cy3 labelled target
strands and C) CFTR MT Cy3 labelled target strands ( indicates increasing target concentration)
27
Figure 10 Representations of the two different direct assay formats investigated in solution phase
gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA MT probe
and were mixed with complementary CFTR WT Cy3 target strands and CFTR MT Cy3 target
strands Hybridization resulted in proximity of gQDs and Cy3 which resulted in FRET 28
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of Cy3
labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol (vii) 75
ix
pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of spots that
may not be visible otherwise 29
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers to
WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash MT target
(D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and Tm) were calculated
using the nearest neighbor method3839 30
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash MT target
(D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and Tm) were calculated
using the nearest neighbor method3839 32
Figure 14 Determination of optimal wash conditions for direct and sandwich assay considered
RG Ratios with variation of formamide concentration for wash times of 0 5 10 15 and 20 min
The optimal wash conditions for direct assay was found to be BB+10F for 5 minutes for (A)
gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal wash conditions for
sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-WT probe sequence and
BB+5F for 20 minutes for (D) gQD-MT probe sequence 38
Figure 15 Concentration-response curves showing the RG ratiometric response of the direct and
sandwich assay formats (Ai) gQD-WT probe conjugates were used for determination of Cy3
labelled WT targets and (Bi) gQD-MT probe conjugates were used for determination of Cy3
labelled MT targets (Ci) gQD-WT probe conjugates were used for determination of unlabelled
WT targets with Cy3 labelled reporters and (Di) gQD-MT probe conjugates were used for
determination of unlabelled MT targets with Cy3 labelled reporters The RG ratiometric response
of the direct assay at the low pmol concentration range was also determined (Aii) gQD-WT probe
conjugates and (Bii) gQD-MT probe conjugates The sandwich assay format (Cii) gQD-WT probe
conjugates and (Dii) gQD-MT probe conjugates Note that the scale for (A) and (B) is logarithmic
Each error bar represents one standard deviation for n=4 replicates 39
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes and
(Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined using
background corrected RG ratio plots for hybridization of gQD-probe conjugates with Cy3 labelled
x
targets (for direct assay A and B) and gQD-probe conjugates with unlabeled targets and Cy3
labelled reporter sequences (for sandwich assay C and D) Response of the hybridization assay
was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-wash (Bi and Di) MT
probe conjugates Post-wash assays yielded signal response shown in Aii and Cii for WT probe
conjugates and in Bii and Dii for MT probe conjugates Error bars represent one standard deviation
for n = 4 replicates 41
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates and
(B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to direct assay
and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was collected for (C)
gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars represent one standard
deviation for n = 4 replicates 42
1
Chapter 1
Introduction
11 Cystic Fibrosis and Genes Associated with Cystic Fibrosis Transmembrane Protein
Cystic fibrosis (CF) is a multi-system fatal autosomal recessive disorder that is
characterized by viscous secretions in the lungs of patients due to mutations in cystic fibrosis
transmembrane conductance regulator protein (CFTR) CF affects 1 in 3000 births with ~70000
people affected worldwide1ndash5 Over 1500 mutations for the CFTR protein have been found but few
are common and fewer result in the disease Of the few mutations responsible for the disease state
the deletion of phenylalanine at the 508 position (∆F508) is responsible for over two-thirds of the
cases while all other mutations account for no more than 5 of the cases individually256
Development of sensing technology for early detection of ∆F508 would serve to enable improved
screening by clinicians to identify the predominant gene carriers The strategies for diagnosing CF
are based on newborn screening (NBS) programs that work via screening for serum markers
including the immunoreactive trypsinogen (IRT) assay7ndash9 This assay is typically followed by
diagnosis of the genetic basis of disease including detection of ∆F508 and related mutations based
on determining the presence of specific oligonucleotide sequences Finally a sweat chloride test
is performed to diagnose patients with CF All of these techniques require skilled technicians to
process samples perform and analyse tests via resource-intensive technologies10 The aim of this
work is to contribute to the development of a low cost easy to use and portable method for sensing
CFTR ∆F508 gene mutations beginning with a focus on a suitable transduction strategy
111 Nucleic Acids based Detection of Genes Associated with CF and ∆F508 mutation of CFTR Gene
There are multiple strategies for transducing the presence of genes associated with CF and
some of the technologies that have been approved by the United Stated Food and Drug
Administration (FDA) for use as in-vitro medical devices are presented in Table 1 (accessed Feb
20th 2018)11
2
Table 1 Summary of FDA approved Nucleic Acid Based Tests for Diagnosis of CF
Manufacturer Trade Name Detection Strategy
Illumina Inc Illumina MiSeqDx Cystic
Fibrosis Clinical Sequencing
Assay
Next-gen sequencing by
synthesis
Illumina MiSeqDx Cystic
Fibrosis 139-Variant Assay
Luminex Molecular
Diagnostics Inc
xTAG Cystic Fibrosis 60 kit v2 Microbead-dye barcode
coupled microarray analysis xTAG Cystic Fibrosis 39 kit v2
Osmetech Molecular
Diagnostics
eSensor CF Genotyping Test Sandwich hybridization assay
with ferrocene tag for cyclic
voltammetry analysis
Nanosphere Inc Verigene CFTR and Verigene
CFTR PolyT Nucleic Acid Tests
Genomic amplification
followed by sandwich assay
with probes and gold
nanoparticle reporters for
analysis
Third Wave Technology Inc InPlex CF Molecular Test PCR coupled with FRET based
microwell plate
Celera Diagnostics Cystic Fibrosis Genotyping
Assay
PCR coupled with capillary
electrophoresis and
oligonucleotide ligation assay
Typically these technologies require the use of specialized facilities and dedicated
technicians for analysis of patient samples and confirmation of CF may take up to a few weeks79
The resources and time required to diagnose patients may be reduced through the development of
point-of-care (POC) devices In particular the use of paper-based test strips with smartphone
detection for on-site rapid screening of disease markers would serve to alleviate the burden placed
on the health care system by more expensive techniques12
At the core of POC technology is the transduction strategy and much effort has gone into
developing optical13 and electrochemical methods14 for generating and measuring signal Yet the
application of this technology has not been investigated for selective sensing of similar nucleic
acid sequences that are often found to be associated with the genetic basis of disease Thus to
further discuss the challenges in this field it is important to address some of the background
technology that has been developed for POC sensors In particular this chapter will discuss nucleic
acid detection and the thermodynamics associated with hybridization interactions the use of
3
formamide as a chaotrope for controlling hybridization the use of nanomaterials like quantum dots
as integrated components in the bioassays for fluorescence resonance energy transfer-based
sensing strategies and the application of paper as a platform and substrate for sensing
12 Nucleic Acids and Oligonucleotide Detection
Deoxyribonucleic acid (DNA) is a class of biopolymers that stores hereditary information
and ribonucleic acid (RNA) functions as the set of instructions for synthesis of proteins15 The two-
step process by which the DNA nucleobase sequence is transcribed for production of RNA and
subsequently RNA is used as a template for translation to produce proteins is referred to as the
central dogma of molecular biology16 Proteins carry out the function that is encoded in the genetic
regions of DNA by interacting with other molecules and biopolymers present within and on the
surface of cells The specific interactions that govern the proteinsrsquo function are due to the three-
dimensional structure of the amino acid sequence that composes proteins17 The order of amino
acids which composes proteins is based on the nucleobase sequence of transcribing RNA (and
therefore DNA) Thus hereditary information stored as the base sequence of DNA can govern the
sequence of amino acids and therefore the structure and function of proteins1617 There are
numerous types of diseases that have arisen due to nucleobase-pair mutations in the sequence of
gene coding regions of DNA18 Mutations of DNA bases influence the amino acid sequence that
compose proteins and a three-base pair deletion like the one found for phenylalanine at codon 508
significantly alters the function of the protein associated with the CFTR gene Other types of
genetic diseases also arise due to mutations of the base pair sequence associated with DNA and
strategies for detection of nucleic acid mutations offer a method to detect the presence of a disease
state
To determine the genetic basis of disease for guiding clinical treatment diagnostic
technology for sensing nucleic acids must be further developed The main goal of clinical
diagnostic technology is to determine the molecular basis of disease for guiding patient therapy
because knowledge obtained from diagnostics are paramount for programing treatment strategies
Clinical diagnostics using a POC strategy offers improved opportunity for wide-scale screening
due to the advantages of low cost ease of manufacturing ease of transport ease of use and ease
of disposal19 One approach to the detection of genetic materials (deoxyribonucleic acid) is based
4
on hybridization and this process requires consideration of the chemical composition structure
and thermodynamics associated with hybridization
121 Structure and Composition of DNA Hybridization
Elucidation of DNArsquos structure and function has a long-storied history that has impacted
many fields of research including chemistry biology and medicine Much of the early work
related to DNA was focused on the structure of DNA with scientists focusing on the key details
related to the chemical composition of the monomers and the structural format of the polymeric
structure of DNA20ndash24 The key findings of the structure of DNA are summarized as follows
1 The structure for the DNA salt is composed of two helical polymer chains that are
coiled around one another and around a shared axis (see Figure 1A) The outside of the
chains is composed of phosphate-sugars groups and the chains are linked together on
the inside via hydrogen bonds between the nucleotide bases
2 The chains are anti-parallel in conformation with ie the 5rsquo end of one chain is bound
via the nucleobases to the 3rsquo end of the other chain
3 Both chains follow a right-handed helix (one type of DNA known as Z-DNA follows
a left-handed helix but this was discovered later)25 and base residues are present on the
chains every 34 Å with structural repeats every ten residues The distance from the
central shared axis to the phosphorous atom is 10 Å
4 The four bases composing DNA bond to a specific pair (see Figure 1B) ie adenine
(purine) binds with thymine (pyrimidine) and guanine (purine) binds with cytosine
(pyrimidine) The relationship of molar equivalency between pairs of bases ie A ndash T
and G ndash C was determined earlier by Chargaff in 195026
Details related to the structure and composition of DNA has formed the basis of our
understanding of the role of DNA in molecular and cell biology Through the structure of DNA
the mechanism for DNA replication27 transcription28 and translation29 for protein synthesis was
elucidated The confirmation of DNA as the storage for hereditary information paved the way for
initiatives like the Human Genome Project and insights from this undertaking have fueled research
regarding the genetic basis of disease30
5
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and
Crick Arrows on the ribbons represent the directionality bias for the single strands and
dimensions for the polymer are presented with one turn of the helix every 34 nm the
distance between base pairs every 034 nm and the distance between the phosphate
backbone and the central axis every 1 nm B shows the hydrogen bonding taking place
between complementary pairs of nucleobases as proposed by Chargaff with adenine (A)
having two hydrogen bonds with thymine (T) and guanine (G) having three hydrogen bonds
with cytosine (C) Image was adapted with permission Copyright Nature Education 201331
122 Thermodynamics of DNA Hybridization
Design and development of DNA-based technologies have been guided by the
thermodynamic modelling of hybridization Techniques like PCR3233 and isothermal
amplification34 rely on accurate control over the annealing of primers and DNA sensors often uses
temperature and chaotropic agents for achieving selectivity35 One of the useful tools for modelling
hybridization and mismatch-based interactions is the nearest neighbor method (NN)36 To explore
the strategy between the NN method and hybridization of DNA it is useful to understand some
details related to predicting the melting temperature (Tm)
First the system of interest will be defined at the equilibrium of dsDNA and ssDNA at the
point where both populations are equal ie half the strands of DNA are in the double helix state
and the other half are single-stranded and are often in various conformations Tm is the temperature
6
at which this equilibrium is found Next the equilibrium constant Keq is defined as being a ratio
of the concentration of dsDNA and ssDNA (as stated in Equation 1) A second expression can be
derived from the Vanrsquot Hoff equation (as stated in Equation 2) where ∆Ho and ∆So are the standard
enthalpy and entropy of hybridization and lnCT is the natural logarithm of the total strand
concentration This second equation can be used to calculate the thermodynamic parameters
related to Tm with the same being true vice versa37
Equation 1 = [][]
Equation 2 = ∆∆
With this foundation investigation into the NN method for modelling can be undertaken
The thermodynamics associated with a base pair are related to some degree with neighboring base
pairs Free energy values and other related parameters have been determined experimentally for
model oligonucleotide sequences This information is then used in conjunction with the nearest
neighbor algorithm (as presented in Equation 3) for obtaining the Tm for the strand of interest
Here base pair doublets are considered for sequence stability with ten unique combinations of
doublets (5rsquo-3rsquo) CG GC AT AA (also = TT) AG (also = CT) AC (also = GT) GA (also = TC)
GG (also = CC) TG (also = CA)38
Equation 3 ∆ = ∆ + ∆ + sum ∆
Equation 4 ∆ = ∆ minus ∆
In Equation 3 the ∆Gi(total) refers to the free energy of the strand of interest ∆G(init)
refers to the free energy of the strand of initiating base pair ∆G(sym) refers to the free energy of
symmetry Gj refers to the free energy associated with one of ten nearest neighbor stacking
interactions and nij is the appearance rate of the stacking interaction of interest Thermodynamic
parameters are also present for entropy and enthalpy allowing for the calculation of the Tm using
Equation 43638 Higher Tm values indicate greater stability than lower Tm values39ndash42 The NN
method can also be used along with a database of mismatch energetics to determine the
thermodynamic parameters related to those sequences
7
Tm values when used in conjunction with the free energies provide a theoretical basis for
designing probe ndash capture strand interactions This understanding can be useful when designing
wash conditions that control stringency for oligonucleotides composed of sequences with high
similarity Stringency control can be achieved using higher temperature (because increasing
temperature results in de-annealing of sequences and has greater effect on hybrids with partial
complementarity)43 by controlling the ionic strength of hybridization44 and via chaotropic agents
such as formamide45 and urea46 Although all strategies are valid for reducing false positive signals
(that arise from partially complementary strands of oligonucleotides) the use of washes containing
chaotropic agents may be more applicable for the POC given that temperature control requires a
temperature module
Chaotropic agents like formamide lower the melting temperature of duplex DNA by
engaging with the hydrogen bond network of DNA The degree by which temperature is lowered
depends on the GC content the conformations of single and duplex forms and the hydration state
of the strand (but typically can range 24 ndash 29 degC per mole of formamide)45 Chemically
formamide strongly associates with DNA is capable of four hydrogen bonds (same as water) and
is a stronger hydrogen bond acceptor than water Formamide ndash water bonds have been reported to
be 20 stronger than water ndash water bonds and it is accepted that formamide engages the hydration
network around DNA4547 Thus using formamide in washes for DNA hybridization can lower the
melting temperature favoring fully complementary hybrids over partially complementary hybrids
123 Notes and Considerations for POC Application
Developing a DNA screening device for the POC application requires consideration of the
many challenges faced by clinicians When screening genetic samples from blood it is important
to note that samples are often complex with proteins and other type of biomolecules (in addition
to cellular debris) and these materials may occlude the signal generated from target detection48
Another challenge to note for nucleic acid-based diagnostics is the low amount of target present in
clinical samples For example one milliliter of human blood contains approximately 107
leukocytes corresponding to femtomolar quantities (fM or attomoles 10-18 moles) of target nucleic
acid Thus detection strategies requiring hybridization-based assay require enzymatic
amplification of the target materials or improved analytical figures of merit for application in
POC49 Presently there are many different strategies for enzymatic amplification of nucleic acids
8
including polymerase chain reaction32 loop-mediated isothermal amplification34 helicase-
dependent isothermal amplification50 and recombinase polymerase amplification51 Post
amplification targets are often detected using hybridization-based assays using Watson-Crick base
pairing for detection of targets of interest Typically capture probes of complementary sequence
to targets are immobilized on a surface and the presence of target forms hybrids that are transduced
via electrochemical5253 or optical strategies54 Nano-surfaces can be integrated into this detection
strategy with oligonucleotides immobilized on the surface of nanoparticles allowing for
transduction via near-field phenomenon
13 Quantum dots
Nanomaterials based on gold and semiconductor composites have had a significant impact
across many different research fields including the chemical physical and biological sciences
Interest in nanoparticles has been driven due to the unique fundamental properties of these
materials as they approach and occupy size regions between bulk material and isolated atoms
Luminescent semiconductor-based quantum dots (QDs) in particular have attracted attention due
to their unique electro-optical properties arising from small size scales (typically ranging from
2 ndash 10 nm and consisting of 102 ndash 104 atoms per crystal) The key factors of interest for these
particles are material composition and size with a combination of the two giving rise to control of
physical properties such as the spectral profile and photon band gap energies55ndash59
There are many strategies for preparing and tuning the electro-optical properties of QDs
but some of the most studied from a synthetic perspective are based on binary composites of
elements from groups II-VI (like CdSe CdS or CdTe) and III-V (like InAs)55ndash58 For binary
composites luminescent properties can be controlled by choice of materials (selecting specific
regions of the UV-vis spectrum) and by control of size whereby smaller crystals are blue-shifted
and larger crystals are red-shifted5759 QDs used in most biological investigations are constructed
in a coreshell manner where the core is composed on a composite of materials previously
mentioned and the shell is composed of an inert coating (ie ZnS see Figure 2A) The QD shell
protects the nanoparticle from environmental degradation forming a protective layer and provides
a larger potential energy barrier for confining the exciton The shell material also provides a
synthetic strategy for controlling the core size and the type of shell allows for designing a class of
ligands for functionalizing the nanoparticle5556
9
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of
ZnS B Quantum dots of different colors are presented with their corresponding core size
image of solution and photoluminescence spectra and color C Diagram representing the
quantum confinement and the change in band gap energy as material size decreases below
the Bohr-exciton radius Here CB and VB represent the conduction and valence band
respectively and Eg represent the band gap energies Image adapted with permission
Copyright 2011 American Chemical Society60
The resulting particles have been incorporated into biological systems using surface ligands
with chemistry that allows the crystals to be stable in aqueous and physiological buffers6162
Further functionalization of these ligands has also allowed for the integration of biomolecules like
nucleic acids63 and proteins64 and polymers like polyethylene glycol (PEG) allowing applications
that extend from biological imaging65 to diagnostic device development and commercial
technologies566667 Optically quantum dots (QD) have broad absorption wavelengths (from the
UV into the visible) narrow and symmetrical emission photoluminescence (PL) profiles (25 nm
of full width at half maximum) high quantum yields and photochemical stability59 These
spectral properties in addition to the large surface area of QDs make them favourable donors for
RET processes
10
131 Quantum Confinement and The Particle in a Box
A brief overview of the quantum mechanics related to QDs will be discussed before
detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids
As the semiconducting material that composes QDs transitions from the bulk scale to the nano-
scale the valence and conductance bands of the semiconductor material split into discrete
energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the
composite of materials however for nanomaterials the band gap can also be tuned by modulating
the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the
dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like
CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an
electron-hole pair When an electron is excited by a photon of greater energy than the band gap
(the probability increases at higher energies yielding broad absorption spectra) the separation of
the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used
to describe this phenomenon is called quantum confinement and the model that best describes it is
the particle in a box575960
In this model a particle is said to be confined in a symmetrical box (of diameter a) where
the center of the box is denoted as = 0 and the edges of the box are denoted as = (
( Here
the potential energy inside the box +( le le
(- is said to be zero and the potential energy outside
the box + le ( ge
(- is said to be infinite The resulting probability of finding a particle outside
the confines of the box is zero 0 = 0 + le ( ge
(-1 and the discrete energy
eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of
interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material
The surface of the material represents the impenetrable barrier (potential energy is infinity)
restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869
As size of the QDs decreases the energy required for excitation increases because the
exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral
properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a
RET based system because the surface area of QDs allows for loading of multiple biomolecules
Thus multiple pathways of RET can take place that can collectively improve energy transfer
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
viii
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society of
Chemistry 2016 16
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A) Reaction
zones consisted of chemically modified paper that were conjugated with gQD-oligonucleotide
probes Zones contained WT and MT controls and test zones where unknown samples were
spotted and imaged Detection was based on the principle of RET with gQDs used as donors and
Cy3 labels on oligonucleotide strands as acceptors (B) Imaging used a smartphone camera with
data processing by ImageJ to split the image to RGB color channels 18
Figure 7 Image of buffer solution leakage from hydrophilic paper zones 23
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines and
emission is shown as solid lines 26
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by fluorescence
spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii) CFTR single
DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT target strands The
concentration-response curves for the different gQD-probe conjugates are shown A WT Cy3
labelled target strands are seen in blue and MT Cy3 labelled target strands are seen in orange
Normalized PL spectra for the calibration curves are shown for B) CFTR WT Cy3 labelled target
strands and C) CFTR MT Cy3 labelled target strands ( indicates increasing target concentration)
27
Figure 10 Representations of the two different direct assay formats investigated in solution phase
gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA MT probe
and were mixed with complementary CFTR WT Cy3 target strands and CFTR MT Cy3 target
strands Hybridization resulted in proximity of gQDs and Cy3 which resulted in FRET 28
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of Cy3
labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol (vii) 75
ix
pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of spots that
may not be visible otherwise 29
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers to
WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash MT target
(D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and Tm) were calculated
using the nearest neighbor method3839 30
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash MT target
(D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and Tm) were calculated
using the nearest neighbor method3839 32
Figure 14 Determination of optimal wash conditions for direct and sandwich assay considered
RG Ratios with variation of formamide concentration for wash times of 0 5 10 15 and 20 min
The optimal wash conditions for direct assay was found to be BB+10F for 5 minutes for (A)
gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal wash conditions for
sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-WT probe sequence and
BB+5F for 20 minutes for (D) gQD-MT probe sequence 38
Figure 15 Concentration-response curves showing the RG ratiometric response of the direct and
sandwich assay formats (Ai) gQD-WT probe conjugates were used for determination of Cy3
labelled WT targets and (Bi) gQD-MT probe conjugates were used for determination of Cy3
labelled MT targets (Ci) gQD-WT probe conjugates were used for determination of unlabelled
WT targets with Cy3 labelled reporters and (Di) gQD-MT probe conjugates were used for
determination of unlabelled MT targets with Cy3 labelled reporters The RG ratiometric response
of the direct assay at the low pmol concentration range was also determined (Aii) gQD-WT probe
conjugates and (Bii) gQD-MT probe conjugates The sandwich assay format (Cii) gQD-WT probe
conjugates and (Dii) gQD-MT probe conjugates Note that the scale for (A) and (B) is logarithmic
Each error bar represents one standard deviation for n=4 replicates 39
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes and
(Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined using
background corrected RG ratio plots for hybridization of gQD-probe conjugates with Cy3 labelled
x
targets (for direct assay A and B) and gQD-probe conjugates with unlabeled targets and Cy3
labelled reporter sequences (for sandwich assay C and D) Response of the hybridization assay
was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-wash (Bi and Di) MT
probe conjugates Post-wash assays yielded signal response shown in Aii and Cii for WT probe
conjugates and in Bii and Dii for MT probe conjugates Error bars represent one standard deviation
for n = 4 replicates 41
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates and
(B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to direct assay
and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was collected for (C)
gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars represent one standard
deviation for n = 4 replicates 42
1
Chapter 1
Introduction
11 Cystic Fibrosis and Genes Associated with Cystic Fibrosis Transmembrane Protein
Cystic fibrosis (CF) is a multi-system fatal autosomal recessive disorder that is
characterized by viscous secretions in the lungs of patients due to mutations in cystic fibrosis
transmembrane conductance regulator protein (CFTR) CF affects 1 in 3000 births with ~70000
people affected worldwide1ndash5 Over 1500 mutations for the CFTR protein have been found but few
are common and fewer result in the disease Of the few mutations responsible for the disease state
the deletion of phenylalanine at the 508 position (∆F508) is responsible for over two-thirds of the
cases while all other mutations account for no more than 5 of the cases individually256
Development of sensing technology for early detection of ∆F508 would serve to enable improved
screening by clinicians to identify the predominant gene carriers The strategies for diagnosing CF
are based on newborn screening (NBS) programs that work via screening for serum markers
including the immunoreactive trypsinogen (IRT) assay7ndash9 This assay is typically followed by
diagnosis of the genetic basis of disease including detection of ∆F508 and related mutations based
on determining the presence of specific oligonucleotide sequences Finally a sweat chloride test
is performed to diagnose patients with CF All of these techniques require skilled technicians to
process samples perform and analyse tests via resource-intensive technologies10 The aim of this
work is to contribute to the development of a low cost easy to use and portable method for sensing
CFTR ∆F508 gene mutations beginning with a focus on a suitable transduction strategy
111 Nucleic Acids based Detection of Genes Associated with CF and ∆F508 mutation of CFTR Gene
There are multiple strategies for transducing the presence of genes associated with CF and
some of the technologies that have been approved by the United Stated Food and Drug
Administration (FDA) for use as in-vitro medical devices are presented in Table 1 (accessed Feb
20th 2018)11
2
Table 1 Summary of FDA approved Nucleic Acid Based Tests for Diagnosis of CF
Manufacturer Trade Name Detection Strategy
Illumina Inc Illumina MiSeqDx Cystic
Fibrosis Clinical Sequencing
Assay
Next-gen sequencing by
synthesis
Illumina MiSeqDx Cystic
Fibrosis 139-Variant Assay
Luminex Molecular
Diagnostics Inc
xTAG Cystic Fibrosis 60 kit v2 Microbead-dye barcode
coupled microarray analysis xTAG Cystic Fibrosis 39 kit v2
Osmetech Molecular
Diagnostics
eSensor CF Genotyping Test Sandwich hybridization assay
with ferrocene tag for cyclic
voltammetry analysis
Nanosphere Inc Verigene CFTR and Verigene
CFTR PolyT Nucleic Acid Tests
Genomic amplification
followed by sandwich assay
with probes and gold
nanoparticle reporters for
analysis
Third Wave Technology Inc InPlex CF Molecular Test PCR coupled with FRET based
microwell plate
Celera Diagnostics Cystic Fibrosis Genotyping
Assay
PCR coupled with capillary
electrophoresis and
oligonucleotide ligation assay
Typically these technologies require the use of specialized facilities and dedicated
technicians for analysis of patient samples and confirmation of CF may take up to a few weeks79
The resources and time required to diagnose patients may be reduced through the development of
point-of-care (POC) devices In particular the use of paper-based test strips with smartphone
detection for on-site rapid screening of disease markers would serve to alleviate the burden placed
on the health care system by more expensive techniques12
At the core of POC technology is the transduction strategy and much effort has gone into
developing optical13 and electrochemical methods14 for generating and measuring signal Yet the
application of this technology has not been investigated for selective sensing of similar nucleic
acid sequences that are often found to be associated with the genetic basis of disease Thus to
further discuss the challenges in this field it is important to address some of the background
technology that has been developed for POC sensors In particular this chapter will discuss nucleic
acid detection and the thermodynamics associated with hybridization interactions the use of
3
formamide as a chaotrope for controlling hybridization the use of nanomaterials like quantum dots
as integrated components in the bioassays for fluorescence resonance energy transfer-based
sensing strategies and the application of paper as a platform and substrate for sensing
12 Nucleic Acids and Oligonucleotide Detection
Deoxyribonucleic acid (DNA) is a class of biopolymers that stores hereditary information
and ribonucleic acid (RNA) functions as the set of instructions for synthesis of proteins15 The two-
step process by which the DNA nucleobase sequence is transcribed for production of RNA and
subsequently RNA is used as a template for translation to produce proteins is referred to as the
central dogma of molecular biology16 Proteins carry out the function that is encoded in the genetic
regions of DNA by interacting with other molecules and biopolymers present within and on the
surface of cells The specific interactions that govern the proteinsrsquo function are due to the three-
dimensional structure of the amino acid sequence that composes proteins17 The order of amino
acids which composes proteins is based on the nucleobase sequence of transcribing RNA (and
therefore DNA) Thus hereditary information stored as the base sequence of DNA can govern the
sequence of amino acids and therefore the structure and function of proteins1617 There are
numerous types of diseases that have arisen due to nucleobase-pair mutations in the sequence of
gene coding regions of DNA18 Mutations of DNA bases influence the amino acid sequence that
compose proteins and a three-base pair deletion like the one found for phenylalanine at codon 508
significantly alters the function of the protein associated with the CFTR gene Other types of
genetic diseases also arise due to mutations of the base pair sequence associated with DNA and
strategies for detection of nucleic acid mutations offer a method to detect the presence of a disease
state
To determine the genetic basis of disease for guiding clinical treatment diagnostic
technology for sensing nucleic acids must be further developed The main goal of clinical
diagnostic technology is to determine the molecular basis of disease for guiding patient therapy
because knowledge obtained from diagnostics are paramount for programing treatment strategies
Clinical diagnostics using a POC strategy offers improved opportunity for wide-scale screening
due to the advantages of low cost ease of manufacturing ease of transport ease of use and ease
of disposal19 One approach to the detection of genetic materials (deoxyribonucleic acid) is based
4
on hybridization and this process requires consideration of the chemical composition structure
and thermodynamics associated with hybridization
121 Structure and Composition of DNA Hybridization
Elucidation of DNArsquos structure and function has a long-storied history that has impacted
many fields of research including chemistry biology and medicine Much of the early work
related to DNA was focused on the structure of DNA with scientists focusing on the key details
related to the chemical composition of the monomers and the structural format of the polymeric
structure of DNA20ndash24 The key findings of the structure of DNA are summarized as follows
1 The structure for the DNA salt is composed of two helical polymer chains that are
coiled around one another and around a shared axis (see Figure 1A) The outside of the
chains is composed of phosphate-sugars groups and the chains are linked together on
the inside via hydrogen bonds between the nucleotide bases
2 The chains are anti-parallel in conformation with ie the 5rsquo end of one chain is bound
via the nucleobases to the 3rsquo end of the other chain
3 Both chains follow a right-handed helix (one type of DNA known as Z-DNA follows
a left-handed helix but this was discovered later)25 and base residues are present on the
chains every 34 Å with structural repeats every ten residues The distance from the
central shared axis to the phosphorous atom is 10 Å
4 The four bases composing DNA bond to a specific pair (see Figure 1B) ie adenine
(purine) binds with thymine (pyrimidine) and guanine (purine) binds with cytosine
(pyrimidine) The relationship of molar equivalency between pairs of bases ie A ndash T
and G ndash C was determined earlier by Chargaff in 195026
Details related to the structure and composition of DNA has formed the basis of our
understanding of the role of DNA in molecular and cell biology Through the structure of DNA
the mechanism for DNA replication27 transcription28 and translation29 for protein synthesis was
elucidated The confirmation of DNA as the storage for hereditary information paved the way for
initiatives like the Human Genome Project and insights from this undertaking have fueled research
regarding the genetic basis of disease30
5
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and
Crick Arrows on the ribbons represent the directionality bias for the single strands and
dimensions for the polymer are presented with one turn of the helix every 34 nm the
distance between base pairs every 034 nm and the distance between the phosphate
backbone and the central axis every 1 nm B shows the hydrogen bonding taking place
between complementary pairs of nucleobases as proposed by Chargaff with adenine (A)
having two hydrogen bonds with thymine (T) and guanine (G) having three hydrogen bonds
with cytosine (C) Image was adapted with permission Copyright Nature Education 201331
122 Thermodynamics of DNA Hybridization
Design and development of DNA-based technologies have been guided by the
thermodynamic modelling of hybridization Techniques like PCR3233 and isothermal
amplification34 rely on accurate control over the annealing of primers and DNA sensors often uses
temperature and chaotropic agents for achieving selectivity35 One of the useful tools for modelling
hybridization and mismatch-based interactions is the nearest neighbor method (NN)36 To explore
the strategy between the NN method and hybridization of DNA it is useful to understand some
details related to predicting the melting temperature (Tm)
First the system of interest will be defined at the equilibrium of dsDNA and ssDNA at the
point where both populations are equal ie half the strands of DNA are in the double helix state
and the other half are single-stranded and are often in various conformations Tm is the temperature
6
at which this equilibrium is found Next the equilibrium constant Keq is defined as being a ratio
of the concentration of dsDNA and ssDNA (as stated in Equation 1) A second expression can be
derived from the Vanrsquot Hoff equation (as stated in Equation 2) where ∆Ho and ∆So are the standard
enthalpy and entropy of hybridization and lnCT is the natural logarithm of the total strand
concentration This second equation can be used to calculate the thermodynamic parameters
related to Tm with the same being true vice versa37
Equation 1 = [][]
Equation 2 = ∆∆
With this foundation investigation into the NN method for modelling can be undertaken
The thermodynamics associated with a base pair are related to some degree with neighboring base
pairs Free energy values and other related parameters have been determined experimentally for
model oligonucleotide sequences This information is then used in conjunction with the nearest
neighbor algorithm (as presented in Equation 3) for obtaining the Tm for the strand of interest
Here base pair doublets are considered for sequence stability with ten unique combinations of
doublets (5rsquo-3rsquo) CG GC AT AA (also = TT) AG (also = CT) AC (also = GT) GA (also = TC)
GG (also = CC) TG (also = CA)38
Equation 3 ∆ = ∆ + ∆ + sum ∆
Equation 4 ∆ = ∆ minus ∆
In Equation 3 the ∆Gi(total) refers to the free energy of the strand of interest ∆G(init)
refers to the free energy of the strand of initiating base pair ∆G(sym) refers to the free energy of
symmetry Gj refers to the free energy associated with one of ten nearest neighbor stacking
interactions and nij is the appearance rate of the stacking interaction of interest Thermodynamic
parameters are also present for entropy and enthalpy allowing for the calculation of the Tm using
Equation 43638 Higher Tm values indicate greater stability than lower Tm values39ndash42 The NN
method can also be used along with a database of mismatch energetics to determine the
thermodynamic parameters related to those sequences
7
Tm values when used in conjunction with the free energies provide a theoretical basis for
designing probe ndash capture strand interactions This understanding can be useful when designing
wash conditions that control stringency for oligonucleotides composed of sequences with high
similarity Stringency control can be achieved using higher temperature (because increasing
temperature results in de-annealing of sequences and has greater effect on hybrids with partial
complementarity)43 by controlling the ionic strength of hybridization44 and via chaotropic agents
such as formamide45 and urea46 Although all strategies are valid for reducing false positive signals
(that arise from partially complementary strands of oligonucleotides) the use of washes containing
chaotropic agents may be more applicable for the POC given that temperature control requires a
temperature module
Chaotropic agents like formamide lower the melting temperature of duplex DNA by
engaging with the hydrogen bond network of DNA The degree by which temperature is lowered
depends on the GC content the conformations of single and duplex forms and the hydration state
of the strand (but typically can range 24 ndash 29 degC per mole of formamide)45 Chemically
formamide strongly associates with DNA is capable of four hydrogen bonds (same as water) and
is a stronger hydrogen bond acceptor than water Formamide ndash water bonds have been reported to
be 20 stronger than water ndash water bonds and it is accepted that formamide engages the hydration
network around DNA4547 Thus using formamide in washes for DNA hybridization can lower the
melting temperature favoring fully complementary hybrids over partially complementary hybrids
123 Notes and Considerations for POC Application
Developing a DNA screening device for the POC application requires consideration of the
many challenges faced by clinicians When screening genetic samples from blood it is important
to note that samples are often complex with proteins and other type of biomolecules (in addition
to cellular debris) and these materials may occlude the signal generated from target detection48
Another challenge to note for nucleic acid-based diagnostics is the low amount of target present in
clinical samples For example one milliliter of human blood contains approximately 107
leukocytes corresponding to femtomolar quantities (fM or attomoles 10-18 moles) of target nucleic
acid Thus detection strategies requiring hybridization-based assay require enzymatic
amplification of the target materials or improved analytical figures of merit for application in
POC49 Presently there are many different strategies for enzymatic amplification of nucleic acids
8
including polymerase chain reaction32 loop-mediated isothermal amplification34 helicase-
dependent isothermal amplification50 and recombinase polymerase amplification51 Post
amplification targets are often detected using hybridization-based assays using Watson-Crick base
pairing for detection of targets of interest Typically capture probes of complementary sequence
to targets are immobilized on a surface and the presence of target forms hybrids that are transduced
via electrochemical5253 or optical strategies54 Nano-surfaces can be integrated into this detection
strategy with oligonucleotides immobilized on the surface of nanoparticles allowing for
transduction via near-field phenomenon
13 Quantum dots
Nanomaterials based on gold and semiconductor composites have had a significant impact
across many different research fields including the chemical physical and biological sciences
Interest in nanoparticles has been driven due to the unique fundamental properties of these
materials as they approach and occupy size regions between bulk material and isolated atoms
Luminescent semiconductor-based quantum dots (QDs) in particular have attracted attention due
to their unique electro-optical properties arising from small size scales (typically ranging from
2 ndash 10 nm and consisting of 102 ndash 104 atoms per crystal) The key factors of interest for these
particles are material composition and size with a combination of the two giving rise to control of
physical properties such as the spectral profile and photon band gap energies55ndash59
There are many strategies for preparing and tuning the electro-optical properties of QDs
but some of the most studied from a synthetic perspective are based on binary composites of
elements from groups II-VI (like CdSe CdS or CdTe) and III-V (like InAs)55ndash58 For binary
composites luminescent properties can be controlled by choice of materials (selecting specific
regions of the UV-vis spectrum) and by control of size whereby smaller crystals are blue-shifted
and larger crystals are red-shifted5759 QDs used in most biological investigations are constructed
in a coreshell manner where the core is composed on a composite of materials previously
mentioned and the shell is composed of an inert coating (ie ZnS see Figure 2A) The QD shell
protects the nanoparticle from environmental degradation forming a protective layer and provides
a larger potential energy barrier for confining the exciton The shell material also provides a
synthetic strategy for controlling the core size and the type of shell allows for designing a class of
ligands for functionalizing the nanoparticle5556
9
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of
ZnS B Quantum dots of different colors are presented with their corresponding core size
image of solution and photoluminescence spectra and color C Diagram representing the
quantum confinement and the change in band gap energy as material size decreases below
the Bohr-exciton radius Here CB and VB represent the conduction and valence band
respectively and Eg represent the band gap energies Image adapted with permission
Copyright 2011 American Chemical Society60
The resulting particles have been incorporated into biological systems using surface ligands
with chemistry that allows the crystals to be stable in aqueous and physiological buffers6162
Further functionalization of these ligands has also allowed for the integration of biomolecules like
nucleic acids63 and proteins64 and polymers like polyethylene glycol (PEG) allowing applications
that extend from biological imaging65 to diagnostic device development and commercial
technologies566667 Optically quantum dots (QD) have broad absorption wavelengths (from the
UV into the visible) narrow and symmetrical emission photoluminescence (PL) profiles (25 nm
of full width at half maximum) high quantum yields and photochemical stability59 These
spectral properties in addition to the large surface area of QDs make them favourable donors for
RET processes
10
131 Quantum Confinement and The Particle in a Box
A brief overview of the quantum mechanics related to QDs will be discussed before
detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids
As the semiconducting material that composes QDs transitions from the bulk scale to the nano-
scale the valence and conductance bands of the semiconductor material split into discrete
energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the
composite of materials however for nanomaterials the band gap can also be tuned by modulating
the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the
dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like
CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an
electron-hole pair When an electron is excited by a photon of greater energy than the band gap
(the probability increases at higher energies yielding broad absorption spectra) the separation of
the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used
to describe this phenomenon is called quantum confinement and the model that best describes it is
the particle in a box575960
In this model a particle is said to be confined in a symmetrical box (of diameter a) where
the center of the box is denoted as = 0 and the edges of the box are denoted as = (
( Here
the potential energy inside the box +( le le
(- is said to be zero and the potential energy outside
the box + le ( ge
(- is said to be infinite The resulting probability of finding a particle outside
the confines of the box is zero 0 = 0 + le ( ge
(-1 and the discrete energy
eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of
interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material
The surface of the material represents the impenetrable barrier (potential energy is infinity)
restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869
As size of the QDs decreases the energy required for excitation increases because the
exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral
properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a
RET based system because the surface area of QDs allows for loading of multiple biomolecules
Thus multiple pathways of RET can take place that can collectively improve energy transfer
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
ix
pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of spots that
may not be visible otherwise 29
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers to
WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash MT target
(D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and Tm) were calculated
using the nearest neighbor method3839 30
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash MT target
(D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and Tm) were calculated
using the nearest neighbor method3839 32
Figure 14 Determination of optimal wash conditions for direct and sandwich assay considered
RG Ratios with variation of formamide concentration for wash times of 0 5 10 15 and 20 min
The optimal wash conditions for direct assay was found to be BB+10F for 5 minutes for (A)
gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal wash conditions for
sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-WT probe sequence and
BB+5F for 20 minutes for (D) gQD-MT probe sequence 38
Figure 15 Concentration-response curves showing the RG ratiometric response of the direct and
sandwich assay formats (Ai) gQD-WT probe conjugates were used for determination of Cy3
labelled WT targets and (Bi) gQD-MT probe conjugates were used for determination of Cy3
labelled MT targets (Ci) gQD-WT probe conjugates were used for determination of unlabelled
WT targets with Cy3 labelled reporters and (Di) gQD-MT probe conjugates were used for
determination of unlabelled MT targets with Cy3 labelled reporters The RG ratiometric response
of the direct assay at the low pmol concentration range was also determined (Aii) gQD-WT probe
conjugates and (Bii) gQD-MT probe conjugates The sandwich assay format (Cii) gQD-WT probe
conjugates and (Dii) gQD-MT probe conjugates Note that the scale for (A) and (B) is logarithmic
Each error bar represents one standard deviation for n=4 replicates 39
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes and
(Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined using
background corrected RG ratio plots for hybridization of gQD-probe conjugates with Cy3 labelled
x
targets (for direct assay A and B) and gQD-probe conjugates with unlabeled targets and Cy3
labelled reporter sequences (for sandwich assay C and D) Response of the hybridization assay
was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-wash (Bi and Di) MT
probe conjugates Post-wash assays yielded signal response shown in Aii and Cii for WT probe
conjugates and in Bii and Dii for MT probe conjugates Error bars represent one standard deviation
for n = 4 replicates 41
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates and
(B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to direct assay
and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was collected for (C)
gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars represent one standard
deviation for n = 4 replicates 42
1
Chapter 1
Introduction
11 Cystic Fibrosis and Genes Associated with Cystic Fibrosis Transmembrane Protein
Cystic fibrosis (CF) is a multi-system fatal autosomal recessive disorder that is
characterized by viscous secretions in the lungs of patients due to mutations in cystic fibrosis
transmembrane conductance regulator protein (CFTR) CF affects 1 in 3000 births with ~70000
people affected worldwide1ndash5 Over 1500 mutations for the CFTR protein have been found but few
are common and fewer result in the disease Of the few mutations responsible for the disease state
the deletion of phenylalanine at the 508 position (∆F508) is responsible for over two-thirds of the
cases while all other mutations account for no more than 5 of the cases individually256
Development of sensing technology for early detection of ∆F508 would serve to enable improved
screening by clinicians to identify the predominant gene carriers The strategies for diagnosing CF
are based on newborn screening (NBS) programs that work via screening for serum markers
including the immunoreactive trypsinogen (IRT) assay7ndash9 This assay is typically followed by
diagnosis of the genetic basis of disease including detection of ∆F508 and related mutations based
on determining the presence of specific oligonucleotide sequences Finally a sweat chloride test
is performed to diagnose patients with CF All of these techniques require skilled technicians to
process samples perform and analyse tests via resource-intensive technologies10 The aim of this
work is to contribute to the development of a low cost easy to use and portable method for sensing
CFTR ∆F508 gene mutations beginning with a focus on a suitable transduction strategy
111 Nucleic Acids based Detection of Genes Associated with CF and ∆F508 mutation of CFTR Gene
There are multiple strategies for transducing the presence of genes associated with CF and
some of the technologies that have been approved by the United Stated Food and Drug
Administration (FDA) for use as in-vitro medical devices are presented in Table 1 (accessed Feb
20th 2018)11
2
Table 1 Summary of FDA approved Nucleic Acid Based Tests for Diagnosis of CF
Manufacturer Trade Name Detection Strategy
Illumina Inc Illumina MiSeqDx Cystic
Fibrosis Clinical Sequencing
Assay
Next-gen sequencing by
synthesis
Illumina MiSeqDx Cystic
Fibrosis 139-Variant Assay
Luminex Molecular
Diagnostics Inc
xTAG Cystic Fibrosis 60 kit v2 Microbead-dye barcode
coupled microarray analysis xTAG Cystic Fibrosis 39 kit v2
Osmetech Molecular
Diagnostics
eSensor CF Genotyping Test Sandwich hybridization assay
with ferrocene tag for cyclic
voltammetry analysis
Nanosphere Inc Verigene CFTR and Verigene
CFTR PolyT Nucleic Acid Tests
Genomic amplification
followed by sandwich assay
with probes and gold
nanoparticle reporters for
analysis
Third Wave Technology Inc InPlex CF Molecular Test PCR coupled with FRET based
microwell plate
Celera Diagnostics Cystic Fibrosis Genotyping
Assay
PCR coupled with capillary
electrophoresis and
oligonucleotide ligation assay
Typically these technologies require the use of specialized facilities and dedicated
technicians for analysis of patient samples and confirmation of CF may take up to a few weeks79
The resources and time required to diagnose patients may be reduced through the development of
point-of-care (POC) devices In particular the use of paper-based test strips with smartphone
detection for on-site rapid screening of disease markers would serve to alleviate the burden placed
on the health care system by more expensive techniques12
At the core of POC technology is the transduction strategy and much effort has gone into
developing optical13 and electrochemical methods14 for generating and measuring signal Yet the
application of this technology has not been investigated for selective sensing of similar nucleic
acid sequences that are often found to be associated with the genetic basis of disease Thus to
further discuss the challenges in this field it is important to address some of the background
technology that has been developed for POC sensors In particular this chapter will discuss nucleic
acid detection and the thermodynamics associated with hybridization interactions the use of
3
formamide as a chaotrope for controlling hybridization the use of nanomaterials like quantum dots
as integrated components in the bioassays for fluorescence resonance energy transfer-based
sensing strategies and the application of paper as a platform and substrate for sensing
12 Nucleic Acids and Oligonucleotide Detection
Deoxyribonucleic acid (DNA) is a class of biopolymers that stores hereditary information
and ribonucleic acid (RNA) functions as the set of instructions for synthesis of proteins15 The two-
step process by which the DNA nucleobase sequence is transcribed for production of RNA and
subsequently RNA is used as a template for translation to produce proteins is referred to as the
central dogma of molecular biology16 Proteins carry out the function that is encoded in the genetic
regions of DNA by interacting with other molecules and biopolymers present within and on the
surface of cells The specific interactions that govern the proteinsrsquo function are due to the three-
dimensional structure of the amino acid sequence that composes proteins17 The order of amino
acids which composes proteins is based on the nucleobase sequence of transcribing RNA (and
therefore DNA) Thus hereditary information stored as the base sequence of DNA can govern the
sequence of amino acids and therefore the structure and function of proteins1617 There are
numerous types of diseases that have arisen due to nucleobase-pair mutations in the sequence of
gene coding regions of DNA18 Mutations of DNA bases influence the amino acid sequence that
compose proteins and a three-base pair deletion like the one found for phenylalanine at codon 508
significantly alters the function of the protein associated with the CFTR gene Other types of
genetic diseases also arise due to mutations of the base pair sequence associated with DNA and
strategies for detection of nucleic acid mutations offer a method to detect the presence of a disease
state
To determine the genetic basis of disease for guiding clinical treatment diagnostic
technology for sensing nucleic acids must be further developed The main goal of clinical
diagnostic technology is to determine the molecular basis of disease for guiding patient therapy
because knowledge obtained from diagnostics are paramount for programing treatment strategies
Clinical diagnostics using a POC strategy offers improved opportunity for wide-scale screening
due to the advantages of low cost ease of manufacturing ease of transport ease of use and ease
of disposal19 One approach to the detection of genetic materials (deoxyribonucleic acid) is based
4
on hybridization and this process requires consideration of the chemical composition structure
and thermodynamics associated with hybridization
121 Structure and Composition of DNA Hybridization
Elucidation of DNArsquos structure and function has a long-storied history that has impacted
many fields of research including chemistry biology and medicine Much of the early work
related to DNA was focused on the structure of DNA with scientists focusing on the key details
related to the chemical composition of the monomers and the structural format of the polymeric
structure of DNA20ndash24 The key findings of the structure of DNA are summarized as follows
1 The structure for the DNA salt is composed of two helical polymer chains that are
coiled around one another and around a shared axis (see Figure 1A) The outside of the
chains is composed of phosphate-sugars groups and the chains are linked together on
the inside via hydrogen bonds between the nucleotide bases
2 The chains are anti-parallel in conformation with ie the 5rsquo end of one chain is bound
via the nucleobases to the 3rsquo end of the other chain
3 Both chains follow a right-handed helix (one type of DNA known as Z-DNA follows
a left-handed helix but this was discovered later)25 and base residues are present on the
chains every 34 Å with structural repeats every ten residues The distance from the
central shared axis to the phosphorous atom is 10 Å
4 The four bases composing DNA bond to a specific pair (see Figure 1B) ie adenine
(purine) binds with thymine (pyrimidine) and guanine (purine) binds with cytosine
(pyrimidine) The relationship of molar equivalency between pairs of bases ie A ndash T
and G ndash C was determined earlier by Chargaff in 195026
Details related to the structure and composition of DNA has formed the basis of our
understanding of the role of DNA in molecular and cell biology Through the structure of DNA
the mechanism for DNA replication27 transcription28 and translation29 for protein synthesis was
elucidated The confirmation of DNA as the storage for hereditary information paved the way for
initiatives like the Human Genome Project and insights from this undertaking have fueled research
regarding the genetic basis of disease30
5
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and
Crick Arrows on the ribbons represent the directionality bias for the single strands and
dimensions for the polymer are presented with one turn of the helix every 34 nm the
distance between base pairs every 034 nm and the distance between the phosphate
backbone and the central axis every 1 nm B shows the hydrogen bonding taking place
between complementary pairs of nucleobases as proposed by Chargaff with adenine (A)
having two hydrogen bonds with thymine (T) and guanine (G) having three hydrogen bonds
with cytosine (C) Image was adapted with permission Copyright Nature Education 201331
122 Thermodynamics of DNA Hybridization
Design and development of DNA-based technologies have been guided by the
thermodynamic modelling of hybridization Techniques like PCR3233 and isothermal
amplification34 rely on accurate control over the annealing of primers and DNA sensors often uses
temperature and chaotropic agents for achieving selectivity35 One of the useful tools for modelling
hybridization and mismatch-based interactions is the nearest neighbor method (NN)36 To explore
the strategy between the NN method and hybridization of DNA it is useful to understand some
details related to predicting the melting temperature (Tm)
First the system of interest will be defined at the equilibrium of dsDNA and ssDNA at the
point where both populations are equal ie half the strands of DNA are in the double helix state
and the other half are single-stranded and are often in various conformations Tm is the temperature
6
at which this equilibrium is found Next the equilibrium constant Keq is defined as being a ratio
of the concentration of dsDNA and ssDNA (as stated in Equation 1) A second expression can be
derived from the Vanrsquot Hoff equation (as stated in Equation 2) where ∆Ho and ∆So are the standard
enthalpy and entropy of hybridization and lnCT is the natural logarithm of the total strand
concentration This second equation can be used to calculate the thermodynamic parameters
related to Tm with the same being true vice versa37
Equation 1 = [][]
Equation 2 = ∆∆
With this foundation investigation into the NN method for modelling can be undertaken
The thermodynamics associated with a base pair are related to some degree with neighboring base
pairs Free energy values and other related parameters have been determined experimentally for
model oligonucleotide sequences This information is then used in conjunction with the nearest
neighbor algorithm (as presented in Equation 3) for obtaining the Tm for the strand of interest
Here base pair doublets are considered for sequence stability with ten unique combinations of
doublets (5rsquo-3rsquo) CG GC AT AA (also = TT) AG (also = CT) AC (also = GT) GA (also = TC)
GG (also = CC) TG (also = CA)38
Equation 3 ∆ = ∆ + ∆ + sum ∆
Equation 4 ∆ = ∆ minus ∆
In Equation 3 the ∆Gi(total) refers to the free energy of the strand of interest ∆G(init)
refers to the free energy of the strand of initiating base pair ∆G(sym) refers to the free energy of
symmetry Gj refers to the free energy associated with one of ten nearest neighbor stacking
interactions and nij is the appearance rate of the stacking interaction of interest Thermodynamic
parameters are also present for entropy and enthalpy allowing for the calculation of the Tm using
Equation 43638 Higher Tm values indicate greater stability than lower Tm values39ndash42 The NN
method can also be used along with a database of mismatch energetics to determine the
thermodynamic parameters related to those sequences
7
Tm values when used in conjunction with the free energies provide a theoretical basis for
designing probe ndash capture strand interactions This understanding can be useful when designing
wash conditions that control stringency for oligonucleotides composed of sequences with high
similarity Stringency control can be achieved using higher temperature (because increasing
temperature results in de-annealing of sequences and has greater effect on hybrids with partial
complementarity)43 by controlling the ionic strength of hybridization44 and via chaotropic agents
such as formamide45 and urea46 Although all strategies are valid for reducing false positive signals
(that arise from partially complementary strands of oligonucleotides) the use of washes containing
chaotropic agents may be more applicable for the POC given that temperature control requires a
temperature module
Chaotropic agents like formamide lower the melting temperature of duplex DNA by
engaging with the hydrogen bond network of DNA The degree by which temperature is lowered
depends on the GC content the conformations of single and duplex forms and the hydration state
of the strand (but typically can range 24 ndash 29 degC per mole of formamide)45 Chemically
formamide strongly associates with DNA is capable of four hydrogen bonds (same as water) and
is a stronger hydrogen bond acceptor than water Formamide ndash water bonds have been reported to
be 20 stronger than water ndash water bonds and it is accepted that formamide engages the hydration
network around DNA4547 Thus using formamide in washes for DNA hybridization can lower the
melting temperature favoring fully complementary hybrids over partially complementary hybrids
123 Notes and Considerations for POC Application
Developing a DNA screening device for the POC application requires consideration of the
many challenges faced by clinicians When screening genetic samples from blood it is important
to note that samples are often complex with proteins and other type of biomolecules (in addition
to cellular debris) and these materials may occlude the signal generated from target detection48
Another challenge to note for nucleic acid-based diagnostics is the low amount of target present in
clinical samples For example one milliliter of human blood contains approximately 107
leukocytes corresponding to femtomolar quantities (fM or attomoles 10-18 moles) of target nucleic
acid Thus detection strategies requiring hybridization-based assay require enzymatic
amplification of the target materials or improved analytical figures of merit for application in
POC49 Presently there are many different strategies for enzymatic amplification of nucleic acids
8
including polymerase chain reaction32 loop-mediated isothermal amplification34 helicase-
dependent isothermal amplification50 and recombinase polymerase amplification51 Post
amplification targets are often detected using hybridization-based assays using Watson-Crick base
pairing for detection of targets of interest Typically capture probes of complementary sequence
to targets are immobilized on a surface and the presence of target forms hybrids that are transduced
via electrochemical5253 or optical strategies54 Nano-surfaces can be integrated into this detection
strategy with oligonucleotides immobilized on the surface of nanoparticles allowing for
transduction via near-field phenomenon
13 Quantum dots
Nanomaterials based on gold and semiconductor composites have had a significant impact
across many different research fields including the chemical physical and biological sciences
Interest in nanoparticles has been driven due to the unique fundamental properties of these
materials as they approach and occupy size regions between bulk material and isolated atoms
Luminescent semiconductor-based quantum dots (QDs) in particular have attracted attention due
to their unique electro-optical properties arising from small size scales (typically ranging from
2 ndash 10 nm and consisting of 102 ndash 104 atoms per crystal) The key factors of interest for these
particles are material composition and size with a combination of the two giving rise to control of
physical properties such as the spectral profile and photon band gap energies55ndash59
There are many strategies for preparing and tuning the electro-optical properties of QDs
but some of the most studied from a synthetic perspective are based on binary composites of
elements from groups II-VI (like CdSe CdS or CdTe) and III-V (like InAs)55ndash58 For binary
composites luminescent properties can be controlled by choice of materials (selecting specific
regions of the UV-vis spectrum) and by control of size whereby smaller crystals are blue-shifted
and larger crystals are red-shifted5759 QDs used in most biological investigations are constructed
in a coreshell manner where the core is composed on a composite of materials previously
mentioned and the shell is composed of an inert coating (ie ZnS see Figure 2A) The QD shell
protects the nanoparticle from environmental degradation forming a protective layer and provides
a larger potential energy barrier for confining the exciton The shell material also provides a
synthetic strategy for controlling the core size and the type of shell allows for designing a class of
ligands for functionalizing the nanoparticle5556
9
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of
ZnS B Quantum dots of different colors are presented with their corresponding core size
image of solution and photoluminescence spectra and color C Diagram representing the
quantum confinement and the change in band gap energy as material size decreases below
the Bohr-exciton radius Here CB and VB represent the conduction and valence band
respectively and Eg represent the band gap energies Image adapted with permission
Copyright 2011 American Chemical Society60
The resulting particles have been incorporated into biological systems using surface ligands
with chemistry that allows the crystals to be stable in aqueous and physiological buffers6162
Further functionalization of these ligands has also allowed for the integration of biomolecules like
nucleic acids63 and proteins64 and polymers like polyethylene glycol (PEG) allowing applications
that extend from biological imaging65 to diagnostic device development and commercial
technologies566667 Optically quantum dots (QD) have broad absorption wavelengths (from the
UV into the visible) narrow and symmetrical emission photoluminescence (PL) profiles (25 nm
of full width at half maximum) high quantum yields and photochemical stability59 These
spectral properties in addition to the large surface area of QDs make them favourable donors for
RET processes
10
131 Quantum Confinement and The Particle in a Box
A brief overview of the quantum mechanics related to QDs will be discussed before
detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids
As the semiconducting material that composes QDs transitions from the bulk scale to the nano-
scale the valence and conductance bands of the semiconductor material split into discrete
energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the
composite of materials however for nanomaterials the band gap can also be tuned by modulating
the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the
dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like
CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an
electron-hole pair When an electron is excited by a photon of greater energy than the band gap
(the probability increases at higher energies yielding broad absorption spectra) the separation of
the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used
to describe this phenomenon is called quantum confinement and the model that best describes it is
the particle in a box575960
In this model a particle is said to be confined in a symmetrical box (of diameter a) where
the center of the box is denoted as = 0 and the edges of the box are denoted as = (
( Here
the potential energy inside the box +( le le
(- is said to be zero and the potential energy outside
the box + le ( ge
(- is said to be infinite The resulting probability of finding a particle outside
the confines of the box is zero 0 = 0 + le ( ge
(-1 and the discrete energy
eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of
interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material
The surface of the material represents the impenetrable barrier (potential energy is infinity)
restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869
As size of the QDs decreases the energy required for excitation increases because the
exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral
properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a
RET based system because the surface area of QDs allows for loading of multiple biomolecules
Thus multiple pathways of RET can take place that can collectively improve energy transfer
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
x
targets (for direct assay A and B) and gQD-probe conjugates with unlabeled targets and Cy3
labelled reporter sequences (for sandwich assay C and D) Response of the hybridization assay
was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-wash (Bi and Di) MT
probe conjugates Post-wash assays yielded signal response shown in Aii and Cii for WT probe
conjugates and in Bii and Dii for MT probe conjugates Error bars represent one standard deviation
for n = 4 replicates 41
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates and
(B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to direct assay
and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was collected for (C)
gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars represent one standard
deviation for n = 4 replicates 42
1
Chapter 1
Introduction
11 Cystic Fibrosis and Genes Associated with Cystic Fibrosis Transmembrane Protein
Cystic fibrosis (CF) is a multi-system fatal autosomal recessive disorder that is
characterized by viscous secretions in the lungs of patients due to mutations in cystic fibrosis
transmembrane conductance regulator protein (CFTR) CF affects 1 in 3000 births with ~70000
people affected worldwide1ndash5 Over 1500 mutations for the CFTR protein have been found but few
are common and fewer result in the disease Of the few mutations responsible for the disease state
the deletion of phenylalanine at the 508 position (∆F508) is responsible for over two-thirds of the
cases while all other mutations account for no more than 5 of the cases individually256
Development of sensing technology for early detection of ∆F508 would serve to enable improved
screening by clinicians to identify the predominant gene carriers The strategies for diagnosing CF
are based on newborn screening (NBS) programs that work via screening for serum markers
including the immunoreactive trypsinogen (IRT) assay7ndash9 This assay is typically followed by
diagnosis of the genetic basis of disease including detection of ∆F508 and related mutations based
on determining the presence of specific oligonucleotide sequences Finally a sweat chloride test
is performed to diagnose patients with CF All of these techniques require skilled technicians to
process samples perform and analyse tests via resource-intensive technologies10 The aim of this
work is to contribute to the development of a low cost easy to use and portable method for sensing
CFTR ∆F508 gene mutations beginning with a focus on a suitable transduction strategy
111 Nucleic Acids based Detection of Genes Associated with CF and ∆F508 mutation of CFTR Gene
There are multiple strategies for transducing the presence of genes associated with CF and
some of the technologies that have been approved by the United Stated Food and Drug
Administration (FDA) for use as in-vitro medical devices are presented in Table 1 (accessed Feb
20th 2018)11
2
Table 1 Summary of FDA approved Nucleic Acid Based Tests for Diagnosis of CF
Manufacturer Trade Name Detection Strategy
Illumina Inc Illumina MiSeqDx Cystic
Fibrosis Clinical Sequencing
Assay
Next-gen sequencing by
synthesis
Illumina MiSeqDx Cystic
Fibrosis 139-Variant Assay
Luminex Molecular
Diagnostics Inc
xTAG Cystic Fibrosis 60 kit v2 Microbead-dye barcode
coupled microarray analysis xTAG Cystic Fibrosis 39 kit v2
Osmetech Molecular
Diagnostics
eSensor CF Genotyping Test Sandwich hybridization assay
with ferrocene tag for cyclic
voltammetry analysis
Nanosphere Inc Verigene CFTR and Verigene
CFTR PolyT Nucleic Acid Tests
Genomic amplification
followed by sandwich assay
with probes and gold
nanoparticle reporters for
analysis
Third Wave Technology Inc InPlex CF Molecular Test PCR coupled with FRET based
microwell plate
Celera Diagnostics Cystic Fibrosis Genotyping
Assay
PCR coupled with capillary
electrophoresis and
oligonucleotide ligation assay
Typically these technologies require the use of specialized facilities and dedicated
technicians for analysis of patient samples and confirmation of CF may take up to a few weeks79
The resources and time required to diagnose patients may be reduced through the development of
point-of-care (POC) devices In particular the use of paper-based test strips with smartphone
detection for on-site rapid screening of disease markers would serve to alleviate the burden placed
on the health care system by more expensive techniques12
At the core of POC technology is the transduction strategy and much effort has gone into
developing optical13 and electrochemical methods14 for generating and measuring signal Yet the
application of this technology has not been investigated for selective sensing of similar nucleic
acid sequences that are often found to be associated with the genetic basis of disease Thus to
further discuss the challenges in this field it is important to address some of the background
technology that has been developed for POC sensors In particular this chapter will discuss nucleic
acid detection and the thermodynamics associated with hybridization interactions the use of
3
formamide as a chaotrope for controlling hybridization the use of nanomaterials like quantum dots
as integrated components in the bioassays for fluorescence resonance energy transfer-based
sensing strategies and the application of paper as a platform and substrate for sensing
12 Nucleic Acids and Oligonucleotide Detection
Deoxyribonucleic acid (DNA) is a class of biopolymers that stores hereditary information
and ribonucleic acid (RNA) functions as the set of instructions for synthesis of proteins15 The two-
step process by which the DNA nucleobase sequence is transcribed for production of RNA and
subsequently RNA is used as a template for translation to produce proteins is referred to as the
central dogma of molecular biology16 Proteins carry out the function that is encoded in the genetic
regions of DNA by interacting with other molecules and biopolymers present within and on the
surface of cells The specific interactions that govern the proteinsrsquo function are due to the three-
dimensional structure of the amino acid sequence that composes proteins17 The order of amino
acids which composes proteins is based on the nucleobase sequence of transcribing RNA (and
therefore DNA) Thus hereditary information stored as the base sequence of DNA can govern the
sequence of amino acids and therefore the structure and function of proteins1617 There are
numerous types of diseases that have arisen due to nucleobase-pair mutations in the sequence of
gene coding regions of DNA18 Mutations of DNA bases influence the amino acid sequence that
compose proteins and a three-base pair deletion like the one found for phenylalanine at codon 508
significantly alters the function of the protein associated with the CFTR gene Other types of
genetic diseases also arise due to mutations of the base pair sequence associated with DNA and
strategies for detection of nucleic acid mutations offer a method to detect the presence of a disease
state
To determine the genetic basis of disease for guiding clinical treatment diagnostic
technology for sensing nucleic acids must be further developed The main goal of clinical
diagnostic technology is to determine the molecular basis of disease for guiding patient therapy
because knowledge obtained from diagnostics are paramount for programing treatment strategies
Clinical diagnostics using a POC strategy offers improved opportunity for wide-scale screening
due to the advantages of low cost ease of manufacturing ease of transport ease of use and ease
of disposal19 One approach to the detection of genetic materials (deoxyribonucleic acid) is based
4
on hybridization and this process requires consideration of the chemical composition structure
and thermodynamics associated with hybridization
121 Structure and Composition of DNA Hybridization
Elucidation of DNArsquos structure and function has a long-storied history that has impacted
many fields of research including chemistry biology and medicine Much of the early work
related to DNA was focused on the structure of DNA with scientists focusing on the key details
related to the chemical composition of the monomers and the structural format of the polymeric
structure of DNA20ndash24 The key findings of the structure of DNA are summarized as follows
1 The structure for the DNA salt is composed of two helical polymer chains that are
coiled around one another and around a shared axis (see Figure 1A) The outside of the
chains is composed of phosphate-sugars groups and the chains are linked together on
the inside via hydrogen bonds between the nucleotide bases
2 The chains are anti-parallel in conformation with ie the 5rsquo end of one chain is bound
via the nucleobases to the 3rsquo end of the other chain
3 Both chains follow a right-handed helix (one type of DNA known as Z-DNA follows
a left-handed helix but this was discovered later)25 and base residues are present on the
chains every 34 Å with structural repeats every ten residues The distance from the
central shared axis to the phosphorous atom is 10 Å
4 The four bases composing DNA bond to a specific pair (see Figure 1B) ie adenine
(purine) binds with thymine (pyrimidine) and guanine (purine) binds with cytosine
(pyrimidine) The relationship of molar equivalency between pairs of bases ie A ndash T
and G ndash C was determined earlier by Chargaff in 195026
Details related to the structure and composition of DNA has formed the basis of our
understanding of the role of DNA in molecular and cell biology Through the structure of DNA
the mechanism for DNA replication27 transcription28 and translation29 for protein synthesis was
elucidated The confirmation of DNA as the storage for hereditary information paved the way for
initiatives like the Human Genome Project and insights from this undertaking have fueled research
regarding the genetic basis of disease30
5
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and
Crick Arrows on the ribbons represent the directionality bias for the single strands and
dimensions for the polymer are presented with one turn of the helix every 34 nm the
distance between base pairs every 034 nm and the distance between the phosphate
backbone and the central axis every 1 nm B shows the hydrogen bonding taking place
between complementary pairs of nucleobases as proposed by Chargaff with adenine (A)
having two hydrogen bonds with thymine (T) and guanine (G) having three hydrogen bonds
with cytosine (C) Image was adapted with permission Copyright Nature Education 201331
122 Thermodynamics of DNA Hybridization
Design and development of DNA-based technologies have been guided by the
thermodynamic modelling of hybridization Techniques like PCR3233 and isothermal
amplification34 rely on accurate control over the annealing of primers and DNA sensors often uses
temperature and chaotropic agents for achieving selectivity35 One of the useful tools for modelling
hybridization and mismatch-based interactions is the nearest neighbor method (NN)36 To explore
the strategy between the NN method and hybridization of DNA it is useful to understand some
details related to predicting the melting temperature (Tm)
First the system of interest will be defined at the equilibrium of dsDNA and ssDNA at the
point where both populations are equal ie half the strands of DNA are in the double helix state
and the other half are single-stranded and are often in various conformations Tm is the temperature
6
at which this equilibrium is found Next the equilibrium constant Keq is defined as being a ratio
of the concentration of dsDNA and ssDNA (as stated in Equation 1) A second expression can be
derived from the Vanrsquot Hoff equation (as stated in Equation 2) where ∆Ho and ∆So are the standard
enthalpy and entropy of hybridization and lnCT is the natural logarithm of the total strand
concentration This second equation can be used to calculate the thermodynamic parameters
related to Tm with the same being true vice versa37
Equation 1 = [][]
Equation 2 = ∆∆
With this foundation investigation into the NN method for modelling can be undertaken
The thermodynamics associated with a base pair are related to some degree with neighboring base
pairs Free energy values and other related parameters have been determined experimentally for
model oligonucleotide sequences This information is then used in conjunction with the nearest
neighbor algorithm (as presented in Equation 3) for obtaining the Tm for the strand of interest
Here base pair doublets are considered for sequence stability with ten unique combinations of
doublets (5rsquo-3rsquo) CG GC AT AA (also = TT) AG (also = CT) AC (also = GT) GA (also = TC)
GG (also = CC) TG (also = CA)38
Equation 3 ∆ = ∆ + ∆ + sum ∆
Equation 4 ∆ = ∆ minus ∆
In Equation 3 the ∆Gi(total) refers to the free energy of the strand of interest ∆G(init)
refers to the free energy of the strand of initiating base pair ∆G(sym) refers to the free energy of
symmetry Gj refers to the free energy associated with one of ten nearest neighbor stacking
interactions and nij is the appearance rate of the stacking interaction of interest Thermodynamic
parameters are also present for entropy and enthalpy allowing for the calculation of the Tm using
Equation 43638 Higher Tm values indicate greater stability than lower Tm values39ndash42 The NN
method can also be used along with a database of mismatch energetics to determine the
thermodynamic parameters related to those sequences
7
Tm values when used in conjunction with the free energies provide a theoretical basis for
designing probe ndash capture strand interactions This understanding can be useful when designing
wash conditions that control stringency for oligonucleotides composed of sequences with high
similarity Stringency control can be achieved using higher temperature (because increasing
temperature results in de-annealing of sequences and has greater effect on hybrids with partial
complementarity)43 by controlling the ionic strength of hybridization44 and via chaotropic agents
such as formamide45 and urea46 Although all strategies are valid for reducing false positive signals
(that arise from partially complementary strands of oligonucleotides) the use of washes containing
chaotropic agents may be more applicable for the POC given that temperature control requires a
temperature module
Chaotropic agents like formamide lower the melting temperature of duplex DNA by
engaging with the hydrogen bond network of DNA The degree by which temperature is lowered
depends on the GC content the conformations of single and duplex forms and the hydration state
of the strand (but typically can range 24 ndash 29 degC per mole of formamide)45 Chemically
formamide strongly associates with DNA is capable of four hydrogen bonds (same as water) and
is a stronger hydrogen bond acceptor than water Formamide ndash water bonds have been reported to
be 20 stronger than water ndash water bonds and it is accepted that formamide engages the hydration
network around DNA4547 Thus using formamide in washes for DNA hybridization can lower the
melting temperature favoring fully complementary hybrids over partially complementary hybrids
123 Notes and Considerations for POC Application
Developing a DNA screening device for the POC application requires consideration of the
many challenges faced by clinicians When screening genetic samples from blood it is important
to note that samples are often complex with proteins and other type of biomolecules (in addition
to cellular debris) and these materials may occlude the signal generated from target detection48
Another challenge to note for nucleic acid-based diagnostics is the low amount of target present in
clinical samples For example one milliliter of human blood contains approximately 107
leukocytes corresponding to femtomolar quantities (fM or attomoles 10-18 moles) of target nucleic
acid Thus detection strategies requiring hybridization-based assay require enzymatic
amplification of the target materials or improved analytical figures of merit for application in
POC49 Presently there are many different strategies for enzymatic amplification of nucleic acids
8
including polymerase chain reaction32 loop-mediated isothermal amplification34 helicase-
dependent isothermal amplification50 and recombinase polymerase amplification51 Post
amplification targets are often detected using hybridization-based assays using Watson-Crick base
pairing for detection of targets of interest Typically capture probes of complementary sequence
to targets are immobilized on a surface and the presence of target forms hybrids that are transduced
via electrochemical5253 or optical strategies54 Nano-surfaces can be integrated into this detection
strategy with oligonucleotides immobilized on the surface of nanoparticles allowing for
transduction via near-field phenomenon
13 Quantum dots
Nanomaterials based on gold and semiconductor composites have had a significant impact
across many different research fields including the chemical physical and biological sciences
Interest in nanoparticles has been driven due to the unique fundamental properties of these
materials as they approach and occupy size regions between bulk material and isolated atoms
Luminescent semiconductor-based quantum dots (QDs) in particular have attracted attention due
to their unique electro-optical properties arising from small size scales (typically ranging from
2 ndash 10 nm and consisting of 102 ndash 104 atoms per crystal) The key factors of interest for these
particles are material composition and size with a combination of the two giving rise to control of
physical properties such as the spectral profile and photon band gap energies55ndash59
There are many strategies for preparing and tuning the electro-optical properties of QDs
but some of the most studied from a synthetic perspective are based on binary composites of
elements from groups II-VI (like CdSe CdS or CdTe) and III-V (like InAs)55ndash58 For binary
composites luminescent properties can be controlled by choice of materials (selecting specific
regions of the UV-vis spectrum) and by control of size whereby smaller crystals are blue-shifted
and larger crystals are red-shifted5759 QDs used in most biological investigations are constructed
in a coreshell manner where the core is composed on a composite of materials previously
mentioned and the shell is composed of an inert coating (ie ZnS see Figure 2A) The QD shell
protects the nanoparticle from environmental degradation forming a protective layer and provides
a larger potential energy barrier for confining the exciton The shell material also provides a
synthetic strategy for controlling the core size and the type of shell allows for designing a class of
ligands for functionalizing the nanoparticle5556
9
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of
ZnS B Quantum dots of different colors are presented with their corresponding core size
image of solution and photoluminescence spectra and color C Diagram representing the
quantum confinement and the change in band gap energy as material size decreases below
the Bohr-exciton radius Here CB and VB represent the conduction and valence band
respectively and Eg represent the band gap energies Image adapted with permission
Copyright 2011 American Chemical Society60
The resulting particles have been incorporated into biological systems using surface ligands
with chemistry that allows the crystals to be stable in aqueous and physiological buffers6162
Further functionalization of these ligands has also allowed for the integration of biomolecules like
nucleic acids63 and proteins64 and polymers like polyethylene glycol (PEG) allowing applications
that extend from biological imaging65 to diagnostic device development and commercial
technologies566667 Optically quantum dots (QD) have broad absorption wavelengths (from the
UV into the visible) narrow and symmetrical emission photoluminescence (PL) profiles (25 nm
of full width at half maximum) high quantum yields and photochemical stability59 These
spectral properties in addition to the large surface area of QDs make them favourable donors for
RET processes
10
131 Quantum Confinement and The Particle in a Box
A brief overview of the quantum mechanics related to QDs will be discussed before
detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids
As the semiconducting material that composes QDs transitions from the bulk scale to the nano-
scale the valence and conductance bands of the semiconductor material split into discrete
energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the
composite of materials however for nanomaterials the band gap can also be tuned by modulating
the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the
dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like
CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an
electron-hole pair When an electron is excited by a photon of greater energy than the band gap
(the probability increases at higher energies yielding broad absorption spectra) the separation of
the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used
to describe this phenomenon is called quantum confinement and the model that best describes it is
the particle in a box575960
In this model a particle is said to be confined in a symmetrical box (of diameter a) where
the center of the box is denoted as = 0 and the edges of the box are denoted as = (
( Here
the potential energy inside the box +( le le
(- is said to be zero and the potential energy outside
the box + le ( ge
(- is said to be infinite The resulting probability of finding a particle outside
the confines of the box is zero 0 = 0 + le ( ge
(-1 and the discrete energy
eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of
interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material
The surface of the material represents the impenetrable barrier (potential energy is infinity)
restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869
As size of the QDs decreases the energy required for excitation increases because the
exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral
properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a
RET based system because the surface area of QDs allows for loading of multiple biomolecules
Thus multiple pathways of RET can take place that can collectively improve energy transfer
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
1
Chapter 1
Introduction
11 Cystic Fibrosis and Genes Associated with Cystic Fibrosis Transmembrane Protein
Cystic fibrosis (CF) is a multi-system fatal autosomal recessive disorder that is
characterized by viscous secretions in the lungs of patients due to mutations in cystic fibrosis
transmembrane conductance regulator protein (CFTR) CF affects 1 in 3000 births with ~70000
people affected worldwide1ndash5 Over 1500 mutations for the CFTR protein have been found but few
are common and fewer result in the disease Of the few mutations responsible for the disease state
the deletion of phenylalanine at the 508 position (∆F508) is responsible for over two-thirds of the
cases while all other mutations account for no more than 5 of the cases individually256
Development of sensing technology for early detection of ∆F508 would serve to enable improved
screening by clinicians to identify the predominant gene carriers The strategies for diagnosing CF
are based on newborn screening (NBS) programs that work via screening for serum markers
including the immunoreactive trypsinogen (IRT) assay7ndash9 This assay is typically followed by
diagnosis of the genetic basis of disease including detection of ∆F508 and related mutations based
on determining the presence of specific oligonucleotide sequences Finally a sweat chloride test
is performed to diagnose patients with CF All of these techniques require skilled technicians to
process samples perform and analyse tests via resource-intensive technologies10 The aim of this
work is to contribute to the development of a low cost easy to use and portable method for sensing
CFTR ∆F508 gene mutations beginning with a focus on a suitable transduction strategy
111 Nucleic Acids based Detection of Genes Associated with CF and ∆F508 mutation of CFTR Gene
There are multiple strategies for transducing the presence of genes associated with CF and
some of the technologies that have been approved by the United Stated Food and Drug
Administration (FDA) for use as in-vitro medical devices are presented in Table 1 (accessed Feb
20th 2018)11
2
Table 1 Summary of FDA approved Nucleic Acid Based Tests for Diagnosis of CF
Manufacturer Trade Name Detection Strategy
Illumina Inc Illumina MiSeqDx Cystic
Fibrosis Clinical Sequencing
Assay
Next-gen sequencing by
synthesis
Illumina MiSeqDx Cystic
Fibrosis 139-Variant Assay
Luminex Molecular
Diagnostics Inc
xTAG Cystic Fibrosis 60 kit v2 Microbead-dye barcode
coupled microarray analysis xTAG Cystic Fibrosis 39 kit v2
Osmetech Molecular
Diagnostics
eSensor CF Genotyping Test Sandwich hybridization assay
with ferrocene tag for cyclic
voltammetry analysis
Nanosphere Inc Verigene CFTR and Verigene
CFTR PolyT Nucleic Acid Tests
Genomic amplification
followed by sandwich assay
with probes and gold
nanoparticle reporters for
analysis
Third Wave Technology Inc InPlex CF Molecular Test PCR coupled with FRET based
microwell plate
Celera Diagnostics Cystic Fibrosis Genotyping
Assay
PCR coupled with capillary
electrophoresis and
oligonucleotide ligation assay
Typically these technologies require the use of specialized facilities and dedicated
technicians for analysis of patient samples and confirmation of CF may take up to a few weeks79
The resources and time required to diagnose patients may be reduced through the development of
point-of-care (POC) devices In particular the use of paper-based test strips with smartphone
detection for on-site rapid screening of disease markers would serve to alleviate the burden placed
on the health care system by more expensive techniques12
At the core of POC technology is the transduction strategy and much effort has gone into
developing optical13 and electrochemical methods14 for generating and measuring signal Yet the
application of this technology has not been investigated for selective sensing of similar nucleic
acid sequences that are often found to be associated with the genetic basis of disease Thus to
further discuss the challenges in this field it is important to address some of the background
technology that has been developed for POC sensors In particular this chapter will discuss nucleic
acid detection and the thermodynamics associated with hybridization interactions the use of
3
formamide as a chaotrope for controlling hybridization the use of nanomaterials like quantum dots
as integrated components in the bioassays for fluorescence resonance energy transfer-based
sensing strategies and the application of paper as a platform and substrate for sensing
12 Nucleic Acids and Oligonucleotide Detection
Deoxyribonucleic acid (DNA) is a class of biopolymers that stores hereditary information
and ribonucleic acid (RNA) functions as the set of instructions for synthesis of proteins15 The two-
step process by which the DNA nucleobase sequence is transcribed for production of RNA and
subsequently RNA is used as a template for translation to produce proteins is referred to as the
central dogma of molecular biology16 Proteins carry out the function that is encoded in the genetic
regions of DNA by interacting with other molecules and biopolymers present within and on the
surface of cells The specific interactions that govern the proteinsrsquo function are due to the three-
dimensional structure of the amino acid sequence that composes proteins17 The order of amino
acids which composes proteins is based on the nucleobase sequence of transcribing RNA (and
therefore DNA) Thus hereditary information stored as the base sequence of DNA can govern the
sequence of amino acids and therefore the structure and function of proteins1617 There are
numerous types of diseases that have arisen due to nucleobase-pair mutations in the sequence of
gene coding regions of DNA18 Mutations of DNA bases influence the amino acid sequence that
compose proteins and a three-base pair deletion like the one found for phenylalanine at codon 508
significantly alters the function of the protein associated with the CFTR gene Other types of
genetic diseases also arise due to mutations of the base pair sequence associated with DNA and
strategies for detection of nucleic acid mutations offer a method to detect the presence of a disease
state
To determine the genetic basis of disease for guiding clinical treatment diagnostic
technology for sensing nucleic acids must be further developed The main goal of clinical
diagnostic technology is to determine the molecular basis of disease for guiding patient therapy
because knowledge obtained from diagnostics are paramount for programing treatment strategies
Clinical diagnostics using a POC strategy offers improved opportunity for wide-scale screening
due to the advantages of low cost ease of manufacturing ease of transport ease of use and ease
of disposal19 One approach to the detection of genetic materials (deoxyribonucleic acid) is based
4
on hybridization and this process requires consideration of the chemical composition structure
and thermodynamics associated with hybridization
121 Structure and Composition of DNA Hybridization
Elucidation of DNArsquos structure and function has a long-storied history that has impacted
many fields of research including chemistry biology and medicine Much of the early work
related to DNA was focused on the structure of DNA with scientists focusing on the key details
related to the chemical composition of the monomers and the structural format of the polymeric
structure of DNA20ndash24 The key findings of the structure of DNA are summarized as follows
1 The structure for the DNA salt is composed of two helical polymer chains that are
coiled around one another and around a shared axis (see Figure 1A) The outside of the
chains is composed of phosphate-sugars groups and the chains are linked together on
the inside via hydrogen bonds between the nucleotide bases
2 The chains are anti-parallel in conformation with ie the 5rsquo end of one chain is bound
via the nucleobases to the 3rsquo end of the other chain
3 Both chains follow a right-handed helix (one type of DNA known as Z-DNA follows
a left-handed helix but this was discovered later)25 and base residues are present on the
chains every 34 Å with structural repeats every ten residues The distance from the
central shared axis to the phosphorous atom is 10 Å
4 The four bases composing DNA bond to a specific pair (see Figure 1B) ie adenine
(purine) binds with thymine (pyrimidine) and guanine (purine) binds with cytosine
(pyrimidine) The relationship of molar equivalency between pairs of bases ie A ndash T
and G ndash C was determined earlier by Chargaff in 195026
Details related to the structure and composition of DNA has formed the basis of our
understanding of the role of DNA in molecular and cell biology Through the structure of DNA
the mechanism for DNA replication27 transcription28 and translation29 for protein synthesis was
elucidated The confirmation of DNA as the storage for hereditary information paved the way for
initiatives like the Human Genome Project and insights from this undertaking have fueled research
regarding the genetic basis of disease30
5
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and
Crick Arrows on the ribbons represent the directionality bias for the single strands and
dimensions for the polymer are presented with one turn of the helix every 34 nm the
distance between base pairs every 034 nm and the distance between the phosphate
backbone and the central axis every 1 nm B shows the hydrogen bonding taking place
between complementary pairs of nucleobases as proposed by Chargaff with adenine (A)
having two hydrogen bonds with thymine (T) and guanine (G) having three hydrogen bonds
with cytosine (C) Image was adapted with permission Copyright Nature Education 201331
122 Thermodynamics of DNA Hybridization
Design and development of DNA-based technologies have been guided by the
thermodynamic modelling of hybridization Techniques like PCR3233 and isothermal
amplification34 rely on accurate control over the annealing of primers and DNA sensors often uses
temperature and chaotropic agents for achieving selectivity35 One of the useful tools for modelling
hybridization and mismatch-based interactions is the nearest neighbor method (NN)36 To explore
the strategy between the NN method and hybridization of DNA it is useful to understand some
details related to predicting the melting temperature (Tm)
First the system of interest will be defined at the equilibrium of dsDNA and ssDNA at the
point where both populations are equal ie half the strands of DNA are in the double helix state
and the other half are single-stranded and are often in various conformations Tm is the temperature
6
at which this equilibrium is found Next the equilibrium constant Keq is defined as being a ratio
of the concentration of dsDNA and ssDNA (as stated in Equation 1) A second expression can be
derived from the Vanrsquot Hoff equation (as stated in Equation 2) where ∆Ho and ∆So are the standard
enthalpy and entropy of hybridization and lnCT is the natural logarithm of the total strand
concentration This second equation can be used to calculate the thermodynamic parameters
related to Tm with the same being true vice versa37
Equation 1 = [][]
Equation 2 = ∆∆
With this foundation investigation into the NN method for modelling can be undertaken
The thermodynamics associated with a base pair are related to some degree with neighboring base
pairs Free energy values and other related parameters have been determined experimentally for
model oligonucleotide sequences This information is then used in conjunction with the nearest
neighbor algorithm (as presented in Equation 3) for obtaining the Tm for the strand of interest
Here base pair doublets are considered for sequence stability with ten unique combinations of
doublets (5rsquo-3rsquo) CG GC AT AA (also = TT) AG (also = CT) AC (also = GT) GA (also = TC)
GG (also = CC) TG (also = CA)38
Equation 3 ∆ = ∆ + ∆ + sum ∆
Equation 4 ∆ = ∆ minus ∆
In Equation 3 the ∆Gi(total) refers to the free energy of the strand of interest ∆G(init)
refers to the free energy of the strand of initiating base pair ∆G(sym) refers to the free energy of
symmetry Gj refers to the free energy associated with one of ten nearest neighbor stacking
interactions and nij is the appearance rate of the stacking interaction of interest Thermodynamic
parameters are also present for entropy and enthalpy allowing for the calculation of the Tm using
Equation 43638 Higher Tm values indicate greater stability than lower Tm values39ndash42 The NN
method can also be used along with a database of mismatch energetics to determine the
thermodynamic parameters related to those sequences
7
Tm values when used in conjunction with the free energies provide a theoretical basis for
designing probe ndash capture strand interactions This understanding can be useful when designing
wash conditions that control stringency for oligonucleotides composed of sequences with high
similarity Stringency control can be achieved using higher temperature (because increasing
temperature results in de-annealing of sequences and has greater effect on hybrids with partial
complementarity)43 by controlling the ionic strength of hybridization44 and via chaotropic agents
such as formamide45 and urea46 Although all strategies are valid for reducing false positive signals
(that arise from partially complementary strands of oligonucleotides) the use of washes containing
chaotropic agents may be more applicable for the POC given that temperature control requires a
temperature module
Chaotropic agents like formamide lower the melting temperature of duplex DNA by
engaging with the hydrogen bond network of DNA The degree by which temperature is lowered
depends on the GC content the conformations of single and duplex forms and the hydration state
of the strand (but typically can range 24 ndash 29 degC per mole of formamide)45 Chemically
formamide strongly associates with DNA is capable of four hydrogen bonds (same as water) and
is a stronger hydrogen bond acceptor than water Formamide ndash water bonds have been reported to
be 20 stronger than water ndash water bonds and it is accepted that formamide engages the hydration
network around DNA4547 Thus using formamide in washes for DNA hybridization can lower the
melting temperature favoring fully complementary hybrids over partially complementary hybrids
123 Notes and Considerations for POC Application
Developing a DNA screening device for the POC application requires consideration of the
many challenges faced by clinicians When screening genetic samples from blood it is important
to note that samples are often complex with proteins and other type of biomolecules (in addition
to cellular debris) and these materials may occlude the signal generated from target detection48
Another challenge to note for nucleic acid-based diagnostics is the low amount of target present in
clinical samples For example one milliliter of human blood contains approximately 107
leukocytes corresponding to femtomolar quantities (fM or attomoles 10-18 moles) of target nucleic
acid Thus detection strategies requiring hybridization-based assay require enzymatic
amplification of the target materials or improved analytical figures of merit for application in
POC49 Presently there are many different strategies for enzymatic amplification of nucleic acids
8
including polymerase chain reaction32 loop-mediated isothermal amplification34 helicase-
dependent isothermal amplification50 and recombinase polymerase amplification51 Post
amplification targets are often detected using hybridization-based assays using Watson-Crick base
pairing for detection of targets of interest Typically capture probes of complementary sequence
to targets are immobilized on a surface and the presence of target forms hybrids that are transduced
via electrochemical5253 or optical strategies54 Nano-surfaces can be integrated into this detection
strategy with oligonucleotides immobilized on the surface of nanoparticles allowing for
transduction via near-field phenomenon
13 Quantum dots
Nanomaterials based on gold and semiconductor composites have had a significant impact
across many different research fields including the chemical physical and biological sciences
Interest in nanoparticles has been driven due to the unique fundamental properties of these
materials as they approach and occupy size regions between bulk material and isolated atoms
Luminescent semiconductor-based quantum dots (QDs) in particular have attracted attention due
to their unique electro-optical properties arising from small size scales (typically ranging from
2 ndash 10 nm and consisting of 102 ndash 104 atoms per crystal) The key factors of interest for these
particles are material composition and size with a combination of the two giving rise to control of
physical properties such as the spectral profile and photon band gap energies55ndash59
There are many strategies for preparing and tuning the electro-optical properties of QDs
but some of the most studied from a synthetic perspective are based on binary composites of
elements from groups II-VI (like CdSe CdS or CdTe) and III-V (like InAs)55ndash58 For binary
composites luminescent properties can be controlled by choice of materials (selecting specific
regions of the UV-vis spectrum) and by control of size whereby smaller crystals are blue-shifted
and larger crystals are red-shifted5759 QDs used in most biological investigations are constructed
in a coreshell manner where the core is composed on a composite of materials previously
mentioned and the shell is composed of an inert coating (ie ZnS see Figure 2A) The QD shell
protects the nanoparticle from environmental degradation forming a protective layer and provides
a larger potential energy barrier for confining the exciton The shell material also provides a
synthetic strategy for controlling the core size and the type of shell allows for designing a class of
ligands for functionalizing the nanoparticle5556
9
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of
ZnS B Quantum dots of different colors are presented with their corresponding core size
image of solution and photoluminescence spectra and color C Diagram representing the
quantum confinement and the change in band gap energy as material size decreases below
the Bohr-exciton radius Here CB and VB represent the conduction and valence band
respectively and Eg represent the band gap energies Image adapted with permission
Copyright 2011 American Chemical Society60
The resulting particles have been incorporated into biological systems using surface ligands
with chemistry that allows the crystals to be stable in aqueous and physiological buffers6162
Further functionalization of these ligands has also allowed for the integration of biomolecules like
nucleic acids63 and proteins64 and polymers like polyethylene glycol (PEG) allowing applications
that extend from biological imaging65 to diagnostic device development and commercial
technologies566667 Optically quantum dots (QD) have broad absorption wavelengths (from the
UV into the visible) narrow and symmetrical emission photoluminescence (PL) profiles (25 nm
of full width at half maximum) high quantum yields and photochemical stability59 These
spectral properties in addition to the large surface area of QDs make them favourable donors for
RET processes
10
131 Quantum Confinement and The Particle in a Box
A brief overview of the quantum mechanics related to QDs will be discussed before
detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids
As the semiconducting material that composes QDs transitions from the bulk scale to the nano-
scale the valence and conductance bands of the semiconductor material split into discrete
energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the
composite of materials however for nanomaterials the band gap can also be tuned by modulating
the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the
dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like
CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an
electron-hole pair When an electron is excited by a photon of greater energy than the band gap
(the probability increases at higher energies yielding broad absorption spectra) the separation of
the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used
to describe this phenomenon is called quantum confinement and the model that best describes it is
the particle in a box575960
In this model a particle is said to be confined in a symmetrical box (of diameter a) where
the center of the box is denoted as = 0 and the edges of the box are denoted as = (
( Here
the potential energy inside the box +( le le
(- is said to be zero and the potential energy outside
the box + le ( ge
(- is said to be infinite The resulting probability of finding a particle outside
the confines of the box is zero 0 = 0 + le ( ge
(-1 and the discrete energy
eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of
interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material
The surface of the material represents the impenetrable barrier (potential energy is infinity)
restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869
As size of the QDs decreases the energy required for excitation increases because the
exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral
properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a
RET based system because the surface area of QDs allows for loading of multiple biomolecules
Thus multiple pathways of RET can take place that can collectively improve energy transfer
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
2
Table 1 Summary of FDA approved Nucleic Acid Based Tests for Diagnosis of CF
Manufacturer Trade Name Detection Strategy
Illumina Inc Illumina MiSeqDx Cystic
Fibrosis Clinical Sequencing
Assay
Next-gen sequencing by
synthesis
Illumina MiSeqDx Cystic
Fibrosis 139-Variant Assay
Luminex Molecular
Diagnostics Inc
xTAG Cystic Fibrosis 60 kit v2 Microbead-dye barcode
coupled microarray analysis xTAG Cystic Fibrosis 39 kit v2
Osmetech Molecular
Diagnostics
eSensor CF Genotyping Test Sandwich hybridization assay
with ferrocene tag for cyclic
voltammetry analysis
Nanosphere Inc Verigene CFTR and Verigene
CFTR PolyT Nucleic Acid Tests
Genomic amplification
followed by sandwich assay
with probes and gold
nanoparticle reporters for
analysis
Third Wave Technology Inc InPlex CF Molecular Test PCR coupled with FRET based
microwell plate
Celera Diagnostics Cystic Fibrosis Genotyping
Assay
PCR coupled with capillary
electrophoresis and
oligonucleotide ligation assay
Typically these technologies require the use of specialized facilities and dedicated
technicians for analysis of patient samples and confirmation of CF may take up to a few weeks79
The resources and time required to diagnose patients may be reduced through the development of
point-of-care (POC) devices In particular the use of paper-based test strips with smartphone
detection for on-site rapid screening of disease markers would serve to alleviate the burden placed
on the health care system by more expensive techniques12
At the core of POC technology is the transduction strategy and much effort has gone into
developing optical13 and electrochemical methods14 for generating and measuring signal Yet the
application of this technology has not been investigated for selective sensing of similar nucleic
acid sequences that are often found to be associated with the genetic basis of disease Thus to
further discuss the challenges in this field it is important to address some of the background
technology that has been developed for POC sensors In particular this chapter will discuss nucleic
acid detection and the thermodynamics associated with hybridization interactions the use of
3
formamide as a chaotrope for controlling hybridization the use of nanomaterials like quantum dots
as integrated components in the bioassays for fluorescence resonance energy transfer-based
sensing strategies and the application of paper as a platform and substrate for sensing
12 Nucleic Acids and Oligonucleotide Detection
Deoxyribonucleic acid (DNA) is a class of biopolymers that stores hereditary information
and ribonucleic acid (RNA) functions as the set of instructions for synthesis of proteins15 The two-
step process by which the DNA nucleobase sequence is transcribed for production of RNA and
subsequently RNA is used as a template for translation to produce proteins is referred to as the
central dogma of molecular biology16 Proteins carry out the function that is encoded in the genetic
regions of DNA by interacting with other molecules and biopolymers present within and on the
surface of cells The specific interactions that govern the proteinsrsquo function are due to the three-
dimensional structure of the amino acid sequence that composes proteins17 The order of amino
acids which composes proteins is based on the nucleobase sequence of transcribing RNA (and
therefore DNA) Thus hereditary information stored as the base sequence of DNA can govern the
sequence of amino acids and therefore the structure and function of proteins1617 There are
numerous types of diseases that have arisen due to nucleobase-pair mutations in the sequence of
gene coding regions of DNA18 Mutations of DNA bases influence the amino acid sequence that
compose proteins and a three-base pair deletion like the one found for phenylalanine at codon 508
significantly alters the function of the protein associated with the CFTR gene Other types of
genetic diseases also arise due to mutations of the base pair sequence associated with DNA and
strategies for detection of nucleic acid mutations offer a method to detect the presence of a disease
state
To determine the genetic basis of disease for guiding clinical treatment diagnostic
technology for sensing nucleic acids must be further developed The main goal of clinical
diagnostic technology is to determine the molecular basis of disease for guiding patient therapy
because knowledge obtained from diagnostics are paramount for programing treatment strategies
Clinical diagnostics using a POC strategy offers improved opportunity for wide-scale screening
due to the advantages of low cost ease of manufacturing ease of transport ease of use and ease
of disposal19 One approach to the detection of genetic materials (deoxyribonucleic acid) is based
4
on hybridization and this process requires consideration of the chemical composition structure
and thermodynamics associated with hybridization
121 Structure and Composition of DNA Hybridization
Elucidation of DNArsquos structure and function has a long-storied history that has impacted
many fields of research including chemistry biology and medicine Much of the early work
related to DNA was focused on the structure of DNA with scientists focusing on the key details
related to the chemical composition of the monomers and the structural format of the polymeric
structure of DNA20ndash24 The key findings of the structure of DNA are summarized as follows
1 The structure for the DNA salt is composed of two helical polymer chains that are
coiled around one another and around a shared axis (see Figure 1A) The outside of the
chains is composed of phosphate-sugars groups and the chains are linked together on
the inside via hydrogen bonds between the nucleotide bases
2 The chains are anti-parallel in conformation with ie the 5rsquo end of one chain is bound
via the nucleobases to the 3rsquo end of the other chain
3 Both chains follow a right-handed helix (one type of DNA known as Z-DNA follows
a left-handed helix but this was discovered later)25 and base residues are present on the
chains every 34 Å with structural repeats every ten residues The distance from the
central shared axis to the phosphorous atom is 10 Å
4 The four bases composing DNA bond to a specific pair (see Figure 1B) ie adenine
(purine) binds with thymine (pyrimidine) and guanine (purine) binds with cytosine
(pyrimidine) The relationship of molar equivalency between pairs of bases ie A ndash T
and G ndash C was determined earlier by Chargaff in 195026
Details related to the structure and composition of DNA has formed the basis of our
understanding of the role of DNA in molecular and cell biology Through the structure of DNA
the mechanism for DNA replication27 transcription28 and translation29 for protein synthesis was
elucidated The confirmation of DNA as the storage for hereditary information paved the way for
initiatives like the Human Genome Project and insights from this undertaking have fueled research
regarding the genetic basis of disease30
5
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and
Crick Arrows on the ribbons represent the directionality bias for the single strands and
dimensions for the polymer are presented with one turn of the helix every 34 nm the
distance between base pairs every 034 nm and the distance between the phosphate
backbone and the central axis every 1 nm B shows the hydrogen bonding taking place
between complementary pairs of nucleobases as proposed by Chargaff with adenine (A)
having two hydrogen bonds with thymine (T) and guanine (G) having three hydrogen bonds
with cytosine (C) Image was adapted with permission Copyright Nature Education 201331
122 Thermodynamics of DNA Hybridization
Design and development of DNA-based technologies have been guided by the
thermodynamic modelling of hybridization Techniques like PCR3233 and isothermal
amplification34 rely on accurate control over the annealing of primers and DNA sensors often uses
temperature and chaotropic agents for achieving selectivity35 One of the useful tools for modelling
hybridization and mismatch-based interactions is the nearest neighbor method (NN)36 To explore
the strategy between the NN method and hybridization of DNA it is useful to understand some
details related to predicting the melting temperature (Tm)
First the system of interest will be defined at the equilibrium of dsDNA and ssDNA at the
point where both populations are equal ie half the strands of DNA are in the double helix state
and the other half are single-stranded and are often in various conformations Tm is the temperature
6
at which this equilibrium is found Next the equilibrium constant Keq is defined as being a ratio
of the concentration of dsDNA and ssDNA (as stated in Equation 1) A second expression can be
derived from the Vanrsquot Hoff equation (as stated in Equation 2) where ∆Ho and ∆So are the standard
enthalpy and entropy of hybridization and lnCT is the natural logarithm of the total strand
concentration This second equation can be used to calculate the thermodynamic parameters
related to Tm with the same being true vice versa37
Equation 1 = [][]
Equation 2 = ∆∆
With this foundation investigation into the NN method for modelling can be undertaken
The thermodynamics associated with a base pair are related to some degree with neighboring base
pairs Free energy values and other related parameters have been determined experimentally for
model oligonucleotide sequences This information is then used in conjunction with the nearest
neighbor algorithm (as presented in Equation 3) for obtaining the Tm for the strand of interest
Here base pair doublets are considered for sequence stability with ten unique combinations of
doublets (5rsquo-3rsquo) CG GC AT AA (also = TT) AG (also = CT) AC (also = GT) GA (also = TC)
GG (also = CC) TG (also = CA)38
Equation 3 ∆ = ∆ + ∆ + sum ∆
Equation 4 ∆ = ∆ minus ∆
In Equation 3 the ∆Gi(total) refers to the free energy of the strand of interest ∆G(init)
refers to the free energy of the strand of initiating base pair ∆G(sym) refers to the free energy of
symmetry Gj refers to the free energy associated with one of ten nearest neighbor stacking
interactions and nij is the appearance rate of the stacking interaction of interest Thermodynamic
parameters are also present for entropy and enthalpy allowing for the calculation of the Tm using
Equation 43638 Higher Tm values indicate greater stability than lower Tm values39ndash42 The NN
method can also be used along with a database of mismatch energetics to determine the
thermodynamic parameters related to those sequences
7
Tm values when used in conjunction with the free energies provide a theoretical basis for
designing probe ndash capture strand interactions This understanding can be useful when designing
wash conditions that control stringency for oligonucleotides composed of sequences with high
similarity Stringency control can be achieved using higher temperature (because increasing
temperature results in de-annealing of sequences and has greater effect on hybrids with partial
complementarity)43 by controlling the ionic strength of hybridization44 and via chaotropic agents
such as formamide45 and urea46 Although all strategies are valid for reducing false positive signals
(that arise from partially complementary strands of oligonucleotides) the use of washes containing
chaotropic agents may be more applicable for the POC given that temperature control requires a
temperature module
Chaotropic agents like formamide lower the melting temperature of duplex DNA by
engaging with the hydrogen bond network of DNA The degree by which temperature is lowered
depends on the GC content the conformations of single and duplex forms and the hydration state
of the strand (but typically can range 24 ndash 29 degC per mole of formamide)45 Chemically
formamide strongly associates with DNA is capable of four hydrogen bonds (same as water) and
is a stronger hydrogen bond acceptor than water Formamide ndash water bonds have been reported to
be 20 stronger than water ndash water bonds and it is accepted that formamide engages the hydration
network around DNA4547 Thus using formamide in washes for DNA hybridization can lower the
melting temperature favoring fully complementary hybrids over partially complementary hybrids
123 Notes and Considerations for POC Application
Developing a DNA screening device for the POC application requires consideration of the
many challenges faced by clinicians When screening genetic samples from blood it is important
to note that samples are often complex with proteins and other type of biomolecules (in addition
to cellular debris) and these materials may occlude the signal generated from target detection48
Another challenge to note for nucleic acid-based diagnostics is the low amount of target present in
clinical samples For example one milliliter of human blood contains approximately 107
leukocytes corresponding to femtomolar quantities (fM or attomoles 10-18 moles) of target nucleic
acid Thus detection strategies requiring hybridization-based assay require enzymatic
amplification of the target materials or improved analytical figures of merit for application in
POC49 Presently there are many different strategies for enzymatic amplification of nucleic acids
8
including polymerase chain reaction32 loop-mediated isothermal amplification34 helicase-
dependent isothermal amplification50 and recombinase polymerase amplification51 Post
amplification targets are often detected using hybridization-based assays using Watson-Crick base
pairing for detection of targets of interest Typically capture probes of complementary sequence
to targets are immobilized on a surface and the presence of target forms hybrids that are transduced
via electrochemical5253 or optical strategies54 Nano-surfaces can be integrated into this detection
strategy with oligonucleotides immobilized on the surface of nanoparticles allowing for
transduction via near-field phenomenon
13 Quantum dots
Nanomaterials based on gold and semiconductor composites have had a significant impact
across many different research fields including the chemical physical and biological sciences
Interest in nanoparticles has been driven due to the unique fundamental properties of these
materials as they approach and occupy size regions between bulk material and isolated atoms
Luminescent semiconductor-based quantum dots (QDs) in particular have attracted attention due
to their unique electro-optical properties arising from small size scales (typically ranging from
2 ndash 10 nm and consisting of 102 ndash 104 atoms per crystal) The key factors of interest for these
particles are material composition and size with a combination of the two giving rise to control of
physical properties such as the spectral profile and photon band gap energies55ndash59
There are many strategies for preparing and tuning the electro-optical properties of QDs
but some of the most studied from a synthetic perspective are based on binary composites of
elements from groups II-VI (like CdSe CdS or CdTe) and III-V (like InAs)55ndash58 For binary
composites luminescent properties can be controlled by choice of materials (selecting specific
regions of the UV-vis spectrum) and by control of size whereby smaller crystals are blue-shifted
and larger crystals are red-shifted5759 QDs used in most biological investigations are constructed
in a coreshell manner where the core is composed on a composite of materials previously
mentioned and the shell is composed of an inert coating (ie ZnS see Figure 2A) The QD shell
protects the nanoparticle from environmental degradation forming a protective layer and provides
a larger potential energy barrier for confining the exciton The shell material also provides a
synthetic strategy for controlling the core size and the type of shell allows for designing a class of
ligands for functionalizing the nanoparticle5556
9
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of
ZnS B Quantum dots of different colors are presented with their corresponding core size
image of solution and photoluminescence spectra and color C Diagram representing the
quantum confinement and the change in band gap energy as material size decreases below
the Bohr-exciton radius Here CB and VB represent the conduction and valence band
respectively and Eg represent the band gap energies Image adapted with permission
Copyright 2011 American Chemical Society60
The resulting particles have been incorporated into biological systems using surface ligands
with chemistry that allows the crystals to be stable in aqueous and physiological buffers6162
Further functionalization of these ligands has also allowed for the integration of biomolecules like
nucleic acids63 and proteins64 and polymers like polyethylene glycol (PEG) allowing applications
that extend from biological imaging65 to diagnostic device development and commercial
technologies566667 Optically quantum dots (QD) have broad absorption wavelengths (from the
UV into the visible) narrow and symmetrical emission photoluminescence (PL) profiles (25 nm
of full width at half maximum) high quantum yields and photochemical stability59 These
spectral properties in addition to the large surface area of QDs make them favourable donors for
RET processes
10
131 Quantum Confinement and The Particle in a Box
A brief overview of the quantum mechanics related to QDs will be discussed before
detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids
As the semiconducting material that composes QDs transitions from the bulk scale to the nano-
scale the valence and conductance bands of the semiconductor material split into discrete
energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the
composite of materials however for nanomaterials the band gap can also be tuned by modulating
the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the
dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like
CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an
electron-hole pair When an electron is excited by a photon of greater energy than the band gap
(the probability increases at higher energies yielding broad absorption spectra) the separation of
the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used
to describe this phenomenon is called quantum confinement and the model that best describes it is
the particle in a box575960
In this model a particle is said to be confined in a symmetrical box (of diameter a) where
the center of the box is denoted as = 0 and the edges of the box are denoted as = (
( Here
the potential energy inside the box +( le le
(- is said to be zero and the potential energy outside
the box + le ( ge
(- is said to be infinite The resulting probability of finding a particle outside
the confines of the box is zero 0 = 0 + le ( ge
(-1 and the discrete energy
eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of
interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material
The surface of the material represents the impenetrable barrier (potential energy is infinity)
restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869
As size of the QDs decreases the energy required for excitation increases because the
exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral
properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a
RET based system because the surface area of QDs allows for loading of multiple biomolecules
Thus multiple pathways of RET can take place that can collectively improve energy transfer
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
3
formamide as a chaotrope for controlling hybridization the use of nanomaterials like quantum dots
as integrated components in the bioassays for fluorescence resonance energy transfer-based
sensing strategies and the application of paper as a platform and substrate for sensing
12 Nucleic Acids and Oligonucleotide Detection
Deoxyribonucleic acid (DNA) is a class of biopolymers that stores hereditary information
and ribonucleic acid (RNA) functions as the set of instructions for synthesis of proteins15 The two-
step process by which the DNA nucleobase sequence is transcribed for production of RNA and
subsequently RNA is used as a template for translation to produce proteins is referred to as the
central dogma of molecular biology16 Proteins carry out the function that is encoded in the genetic
regions of DNA by interacting with other molecules and biopolymers present within and on the
surface of cells The specific interactions that govern the proteinsrsquo function are due to the three-
dimensional structure of the amino acid sequence that composes proteins17 The order of amino
acids which composes proteins is based on the nucleobase sequence of transcribing RNA (and
therefore DNA) Thus hereditary information stored as the base sequence of DNA can govern the
sequence of amino acids and therefore the structure and function of proteins1617 There are
numerous types of diseases that have arisen due to nucleobase-pair mutations in the sequence of
gene coding regions of DNA18 Mutations of DNA bases influence the amino acid sequence that
compose proteins and a three-base pair deletion like the one found for phenylalanine at codon 508
significantly alters the function of the protein associated with the CFTR gene Other types of
genetic diseases also arise due to mutations of the base pair sequence associated with DNA and
strategies for detection of nucleic acid mutations offer a method to detect the presence of a disease
state
To determine the genetic basis of disease for guiding clinical treatment diagnostic
technology for sensing nucleic acids must be further developed The main goal of clinical
diagnostic technology is to determine the molecular basis of disease for guiding patient therapy
because knowledge obtained from diagnostics are paramount for programing treatment strategies
Clinical diagnostics using a POC strategy offers improved opportunity for wide-scale screening
due to the advantages of low cost ease of manufacturing ease of transport ease of use and ease
of disposal19 One approach to the detection of genetic materials (deoxyribonucleic acid) is based
4
on hybridization and this process requires consideration of the chemical composition structure
and thermodynamics associated with hybridization
121 Structure and Composition of DNA Hybridization
Elucidation of DNArsquos structure and function has a long-storied history that has impacted
many fields of research including chemistry biology and medicine Much of the early work
related to DNA was focused on the structure of DNA with scientists focusing on the key details
related to the chemical composition of the monomers and the structural format of the polymeric
structure of DNA20ndash24 The key findings of the structure of DNA are summarized as follows
1 The structure for the DNA salt is composed of two helical polymer chains that are
coiled around one another and around a shared axis (see Figure 1A) The outside of the
chains is composed of phosphate-sugars groups and the chains are linked together on
the inside via hydrogen bonds between the nucleotide bases
2 The chains are anti-parallel in conformation with ie the 5rsquo end of one chain is bound
via the nucleobases to the 3rsquo end of the other chain
3 Both chains follow a right-handed helix (one type of DNA known as Z-DNA follows
a left-handed helix but this was discovered later)25 and base residues are present on the
chains every 34 Å with structural repeats every ten residues The distance from the
central shared axis to the phosphorous atom is 10 Å
4 The four bases composing DNA bond to a specific pair (see Figure 1B) ie adenine
(purine) binds with thymine (pyrimidine) and guanine (purine) binds with cytosine
(pyrimidine) The relationship of molar equivalency between pairs of bases ie A ndash T
and G ndash C was determined earlier by Chargaff in 195026
Details related to the structure and composition of DNA has formed the basis of our
understanding of the role of DNA in molecular and cell biology Through the structure of DNA
the mechanism for DNA replication27 transcription28 and translation29 for protein synthesis was
elucidated The confirmation of DNA as the storage for hereditary information paved the way for
initiatives like the Human Genome Project and insights from this undertaking have fueled research
regarding the genetic basis of disease30
5
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and
Crick Arrows on the ribbons represent the directionality bias for the single strands and
dimensions for the polymer are presented with one turn of the helix every 34 nm the
distance between base pairs every 034 nm and the distance between the phosphate
backbone and the central axis every 1 nm B shows the hydrogen bonding taking place
between complementary pairs of nucleobases as proposed by Chargaff with adenine (A)
having two hydrogen bonds with thymine (T) and guanine (G) having three hydrogen bonds
with cytosine (C) Image was adapted with permission Copyright Nature Education 201331
122 Thermodynamics of DNA Hybridization
Design and development of DNA-based technologies have been guided by the
thermodynamic modelling of hybridization Techniques like PCR3233 and isothermal
amplification34 rely on accurate control over the annealing of primers and DNA sensors often uses
temperature and chaotropic agents for achieving selectivity35 One of the useful tools for modelling
hybridization and mismatch-based interactions is the nearest neighbor method (NN)36 To explore
the strategy between the NN method and hybridization of DNA it is useful to understand some
details related to predicting the melting temperature (Tm)
First the system of interest will be defined at the equilibrium of dsDNA and ssDNA at the
point where both populations are equal ie half the strands of DNA are in the double helix state
and the other half are single-stranded and are often in various conformations Tm is the temperature
6
at which this equilibrium is found Next the equilibrium constant Keq is defined as being a ratio
of the concentration of dsDNA and ssDNA (as stated in Equation 1) A second expression can be
derived from the Vanrsquot Hoff equation (as stated in Equation 2) where ∆Ho and ∆So are the standard
enthalpy and entropy of hybridization and lnCT is the natural logarithm of the total strand
concentration This second equation can be used to calculate the thermodynamic parameters
related to Tm with the same being true vice versa37
Equation 1 = [][]
Equation 2 = ∆∆
With this foundation investigation into the NN method for modelling can be undertaken
The thermodynamics associated with a base pair are related to some degree with neighboring base
pairs Free energy values and other related parameters have been determined experimentally for
model oligonucleotide sequences This information is then used in conjunction with the nearest
neighbor algorithm (as presented in Equation 3) for obtaining the Tm for the strand of interest
Here base pair doublets are considered for sequence stability with ten unique combinations of
doublets (5rsquo-3rsquo) CG GC AT AA (also = TT) AG (also = CT) AC (also = GT) GA (also = TC)
GG (also = CC) TG (also = CA)38
Equation 3 ∆ = ∆ + ∆ + sum ∆
Equation 4 ∆ = ∆ minus ∆
In Equation 3 the ∆Gi(total) refers to the free energy of the strand of interest ∆G(init)
refers to the free energy of the strand of initiating base pair ∆G(sym) refers to the free energy of
symmetry Gj refers to the free energy associated with one of ten nearest neighbor stacking
interactions and nij is the appearance rate of the stacking interaction of interest Thermodynamic
parameters are also present for entropy and enthalpy allowing for the calculation of the Tm using
Equation 43638 Higher Tm values indicate greater stability than lower Tm values39ndash42 The NN
method can also be used along with a database of mismatch energetics to determine the
thermodynamic parameters related to those sequences
7
Tm values when used in conjunction with the free energies provide a theoretical basis for
designing probe ndash capture strand interactions This understanding can be useful when designing
wash conditions that control stringency for oligonucleotides composed of sequences with high
similarity Stringency control can be achieved using higher temperature (because increasing
temperature results in de-annealing of sequences and has greater effect on hybrids with partial
complementarity)43 by controlling the ionic strength of hybridization44 and via chaotropic agents
such as formamide45 and urea46 Although all strategies are valid for reducing false positive signals
(that arise from partially complementary strands of oligonucleotides) the use of washes containing
chaotropic agents may be more applicable for the POC given that temperature control requires a
temperature module
Chaotropic agents like formamide lower the melting temperature of duplex DNA by
engaging with the hydrogen bond network of DNA The degree by which temperature is lowered
depends on the GC content the conformations of single and duplex forms and the hydration state
of the strand (but typically can range 24 ndash 29 degC per mole of formamide)45 Chemically
formamide strongly associates with DNA is capable of four hydrogen bonds (same as water) and
is a stronger hydrogen bond acceptor than water Formamide ndash water bonds have been reported to
be 20 stronger than water ndash water bonds and it is accepted that formamide engages the hydration
network around DNA4547 Thus using formamide in washes for DNA hybridization can lower the
melting temperature favoring fully complementary hybrids over partially complementary hybrids
123 Notes and Considerations for POC Application
Developing a DNA screening device for the POC application requires consideration of the
many challenges faced by clinicians When screening genetic samples from blood it is important
to note that samples are often complex with proteins and other type of biomolecules (in addition
to cellular debris) and these materials may occlude the signal generated from target detection48
Another challenge to note for nucleic acid-based diagnostics is the low amount of target present in
clinical samples For example one milliliter of human blood contains approximately 107
leukocytes corresponding to femtomolar quantities (fM or attomoles 10-18 moles) of target nucleic
acid Thus detection strategies requiring hybridization-based assay require enzymatic
amplification of the target materials or improved analytical figures of merit for application in
POC49 Presently there are many different strategies for enzymatic amplification of nucleic acids
8
including polymerase chain reaction32 loop-mediated isothermal amplification34 helicase-
dependent isothermal amplification50 and recombinase polymerase amplification51 Post
amplification targets are often detected using hybridization-based assays using Watson-Crick base
pairing for detection of targets of interest Typically capture probes of complementary sequence
to targets are immobilized on a surface and the presence of target forms hybrids that are transduced
via electrochemical5253 or optical strategies54 Nano-surfaces can be integrated into this detection
strategy with oligonucleotides immobilized on the surface of nanoparticles allowing for
transduction via near-field phenomenon
13 Quantum dots
Nanomaterials based on gold and semiconductor composites have had a significant impact
across many different research fields including the chemical physical and biological sciences
Interest in nanoparticles has been driven due to the unique fundamental properties of these
materials as they approach and occupy size regions between bulk material and isolated atoms
Luminescent semiconductor-based quantum dots (QDs) in particular have attracted attention due
to their unique electro-optical properties arising from small size scales (typically ranging from
2 ndash 10 nm and consisting of 102 ndash 104 atoms per crystal) The key factors of interest for these
particles are material composition and size with a combination of the two giving rise to control of
physical properties such as the spectral profile and photon band gap energies55ndash59
There are many strategies for preparing and tuning the electro-optical properties of QDs
but some of the most studied from a synthetic perspective are based on binary composites of
elements from groups II-VI (like CdSe CdS or CdTe) and III-V (like InAs)55ndash58 For binary
composites luminescent properties can be controlled by choice of materials (selecting specific
regions of the UV-vis spectrum) and by control of size whereby smaller crystals are blue-shifted
and larger crystals are red-shifted5759 QDs used in most biological investigations are constructed
in a coreshell manner where the core is composed on a composite of materials previously
mentioned and the shell is composed of an inert coating (ie ZnS see Figure 2A) The QD shell
protects the nanoparticle from environmental degradation forming a protective layer and provides
a larger potential energy barrier for confining the exciton The shell material also provides a
synthetic strategy for controlling the core size and the type of shell allows for designing a class of
ligands for functionalizing the nanoparticle5556
9
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of
ZnS B Quantum dots of different colors are presented with their corresponding core size
image of solution and photoluminescence spectra and color C Diagram representing the
quantum confinement and the change in band gap energy as material size decreases below
the Bohr-exciton radius Here CB and VB represent the conduction and valence band
respectively and Eg represent the band gap energies Image adapted with permission
Copyright 2011 American Chemical Society60
The resulting particles have been incorporated into biological systems using surface ligands
with chemistry that allows the crystals to be stable in aqueous and physiological buffers6162
Further functionalization of these ligands has also allowed for the integration of biomolecules like
nucleic acids63 and proteins64 and polymers like polyethylene glycol (PEG) allowing applications
that extend from biological imaging65 to diagnostic device development and commercial
technologies566667 Optically quantum dots (QD) have broad absorption wavelengths (from the
UV into the visible) narrow and symmetrical emission photoluminescence (PL) profiles (25 nm
of full width at half maximum) high quantum yields and photochemical stability59 These
spectral properties in addition to the large surface area of QDs make them favourable donors for
RET processes
10
131 Quantum Confinement and The Particle in a Box
A brief overview of the quantum mechanics related to QDs will be discussed before
detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids
As the semiconducting material that composes QDs transitions from the bulk scale to the nano-
scale the valence and conductance bands of the semiconductor material split into discrete
energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the
composite of materials however for nanomaterials the band gap can also be tuned by modulating
the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the
dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like
CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an
electron-hole pair When an electron is excited by a photon of greater energy than the band gap
(the probability increases at higher energies yielding broad absorption spectra) the separation of
the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used
to describe this phenomenon is called quantum confinement and the model that best describes it is
the particle in a box575960
In this model a particle is said to be confined in a symmetrical box (of diameter a) where
the center of the box is denoted as = 0 and the edges of the box are denoted as = (
( Here
the potential energy inside the box +( le le
(- is said to be zero and the potential energy outside
the box + le ( ge
(- is said to be infinite The resulting probability of finding a particle outside
the confines of the box is zero 0 = 0 + le ( ge
(-1 and the discrete energy
eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of
interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material
The surface of the material represents the impenetrable barrier (potential energy is infinity)
restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869
As size of the QDs decreases the energy required for excitation increases because the
exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral
properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a
RET based system because the surface area of QDs allows for loading of multiple biomolecules
Thus multiple pathways of RET can take place that can collectively improve energy transfer
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
4
on hybridization and this process requires consideration of the chemical composition structure
and thermodynamics associated with hybridization
121 Structure and Composition of DNA Hybridization
Elucidation of DNArsquos structure and function has a long-storied history that has impacted
many fields of research including chemistry biology and medicine Much of the early work
related to DNA was focused on the structure of DNA with scientists focusing on the key details
related to the chemical composition of the monomers and the structural format of the polymeric
structure of DNA20ndash24 The key findings of the structure of DNA are summarized as follows
1 The structure for the DNA salt is composed of two helical polymer chains that are
coiled around one another and around a shared axis (see Figure 1A) The outside of the
chains is composed of phosphate-sugars groups and the chains are linked together on
the inside via hydrogen bonds between the nucleotide bases
2 The chains are anti-parallel in conformation with ie the 5rsquo end of one chain is bound
via the nucleobases to the 3rsquo end of the other chain
3 Both chains follow a right-handed helix (one type of DNA known as Z-DNA follows
a left-handed helix but this was discovered later)25 and base residues are present on the
chains every 34 Å with structural repeats every ten residues The distance from the
central shared axis to the phosphorous atom is 10 Å
4 The four bases composing DNA bond to a specific pair (see Figure 1B) ie adenine
(purine) binds with thymine (pyrimidine) and guanine (purine) binds with cytosine
(pyrimidine) The relationship of molar equivalency between pairs of bases ie A ndash T
and G ndash C was determined earlier by Chargaff in 195026
Details related to the structure and composition of DNA has formed the basis of our
understanding of the role of DNA in molecular and cell biology Through the structure of DNA
the mechanism for DNA replication27 transcription28 and translation29 for protein synthesis was
elucidated The confirmation of DNA as the storage for hereditary information paved the way for
initiatives like the Human Genome Project and insights from this undertaking have fueled research
regarding the genetic basis of disease30
5
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and
Crick Arrows on the ribbons represent the directionality bias for the single strands and
dimensions for the polymer are presented with one turn of the helix every 34 nm the
distance between base pairs every 034 nm and the distance between the phosphate
backbone and the central axis every 1 nm B shows the hydrogen bonding taking place
between complementary pairs of nucleobases as proposed by Chargaff with adenine (A)
having two hydrogen bonds with thymine (T) and guanine (G) having three hydrogen bonds
with cytosine (C) Image was adapted with permission Copyright Nature Education 201331
122 Thermodynamics of DNA Hybridization
Design and development of DNA-based technologies have been guided by the
thermodynamic modelling of hybridization Techniques like PCR3233 and isothermal
amplification34 rely on accurate control over the annealing of primers and DNA sensors often uses
temperature and chaotropic agents for achieving selectivity35 One of the useful tools for modelling
hybridization and mismatch-based interactions is the nearest neighbor method (NN)36 To explore
the strategy between the NN method and hybridization of DNA it is useful to understand some
details related to predicting the melting temperature (Tm)
First the system of interest will be defined at the equilibrium of dsDNA and ssDNA at the
point where both populations are equal ie half the strands of DNA are in the double helix state
and the other half are single-stranded and are often in various conformations Tm is the temperature
6
at which this equilibrium is found Next the equilibrium constant Keq is defined as being a ratio
of the concentration of dsDNA and ssDNA (as stated in Equation 1) A second expression can be
derived from the Vanrsquot Hoff equation (as stated in Equation 2) where ∆Ho and ∆So are the standard
enthalpy and entropy of hybridization and lnCT is the natural logarithm of the total strand
concentration This second equation can be used to calculate the thermodynamic parameters
related to Tm with the same being true vice versa37
Equation 1 = [][]
Equation 2 = ∆∆
With this foundation investigation into the NN method for modelling can be undertaken
The thermodynamics associated with a base pair are related to some degree with neighboring base
pairs Free energy values and other related parameters have been determined experimentally for
model oligonucleotide sequences This information is then used in conjunction with the nearest
neighbor algorithm (as presented in Equation 3) for obtaining the Tm for the strand of interest
Here base pair doublets are considered for sequence stability with ten unique combinations of
doublets (5rsquo-3rsquo) CG GC AT AA (also = TT) AG (also = CT) AC (also = GT) GA (also = TC)
GG (also = CC) TG (also = CA)38
Equation 3 ∆ = ∆ + ∆ + sum ∆
Equation 4 ∆ = ∆ minus ∆
In Equation 3 the ∆Gi(total) refers to the free energy of the strand of interest ∆G(init)
refers to the free energy of the strand of initiating base pair ∆G(sym) refers to the free energy of
symmetry Gj refers to the free energy associated with one of ten nearest neighbor stacking
interactions and nij is the appearance rate of the stacking interaction of interest Thermodynamic
parameters are also present for entropy and enthalpy allowing for the calculation of the Tm using
Equation 43638 Higher Tm values indicate greater stability than lower Tm values39ndash42 The NN
method can also be used along with a database of mismatch energetics to determine the
thermodynamic parameters related to those sequences
7
Tm values when used in conjunction with the free energies provide a theoretical basis for
designing probe ndash capture strand interactions This understanding can be useful when designing
wash conditions that control stringency for oligonucleotides composed of sequences with high
similarity Stringency control can be achieved using higher temperature (because increasing
temperature results in de-annealing of sequences and has greater effect on hybrids with partial
complementarity)43 by controlling the ionic strength of hybridization44 and via chaotropic agents
such as formamide45 and urea46 Although all strategies are valid for reducing false positive signals
(that arise from partially complementary strands of oligonucleotides) the use of washes containing
chaotropic agents may be more applicable for the POC given that temperature control requires a
temperature module
Chaotropic agents like formamide lower the melting temperature of duplex DNA by
engaging with the hydrogen bond network of DNA The degree by which temperature is lowered
depends on the GC content the conformations of single and duplex forms and the hydration state
of the strand (but typically can range 24 ndash 29 degC per mole of formamide)45 Chemically
formamide strongly associates with DNA is capable of four hydrogen bonds (same as water) and
is a stronger hydrogen bond acceptor than water Formamide ndash water bonds have been reported to
be 20 stronger than water ndash water bonds and it is accepted that formamide engages the hydration
network around DNA4547 Thus using formamide in washes for DNA hybridization can lower the
melting temperature favoring fully complementary hybrids over partially complementary hybrids
123 Notes and Considerations for POC Application
Developing a DNA screening device for the POC application requires consideration of the
many challenges faced by clinicians When screening genetic samples from blood it is important
to note that samples are often complex with proteins and other type of biomolecules (in addition
to cellular debris) and these materials may occlude the signal generated from target detection48
Another challenge to note for nucleic acid-based diagnostics is the low amount of target present in
clinical samples For example one milliliter of human blood contains approximately 107
leukocytes corresponding to femtomolar quantities (fM or attomoles 10-18 moles) of target nucleic
acid Thus detection strategies requiring hybridization-based assay require enzymatic
amplification of the target materials or improved analytical figures of merit for application in
POC49 Presently there are many different strategies for enzymatic amplification of nucleic acids
8
including polymerase chain reaction32 loop-mediated isothermal amplification34 helicase-
dependent isothermal amplification50 and recombinase polymerase amplification51 Post
amplification targets are often detected using hybridization-based assays using Watson-Crick base
pairing for detection of targets of interest Typically capture probes of complementary sequence
to targets are immobilized on a surface and the presence of target forms hybrids that are transduced
via electrochemical5253 or optical strategies54 Nano-surfaces can be integrated into this detection
strategy with oligonucleotides immobilized on the surface of nanoparticles allowing for
transduction via near-field phenomenon
13 Quantum dots
Nanomaterials based on gold and semiconductor composites have had a significant impact
across many different research fields including the chemical physical and biological sciences
Interest in nanoparticles has been driven due to the unique fundamental properties of these
materials as they approach and occupy size regions between bulk material and isolated atoms
Luminescent semiconductor-based quantum dots (QDs) in particular have attracted attention due
to their unique electro-optical properties arising from small size scales (typically ranging from
2 ndash 10 nm and consisting of 102 ndash 104 atoms per crystal) The key factors of interest for these
particles are material composition and size with a combination of the two giving rise to control of
physical properties such as the spectral profile and photon band gap energies55ndash59
There are many strategies for preparing and tuning the electro-optical properties of QDs
but some of the most studied from a synthetic perspective are based on binary composites of
elements from groups II-VI (like CdSe CdS or CdTe) and III-V (like InAs)55ndash58 For binary
composites luminescent properties can be controlled by choice of materials (selecting specific
regions of the UV-vis spectrum) and by control of size whereby smaller crystals are blue-shifted
and larger crystals are red-shifted5759 QDs used in most biological investigations are constructed
in a coreshell manner where the core is composed on a composite of materials previously
mentioned and the shell is composed of an inert coating (ie ZnS see Figure 2A) The QD shell
protects the nanoparticle from environmental degradation forming a protective layer and provides
a larger potential energy barrier for confining the exciton The shell material also provides a
synthetic strategy for controlling the core size and the type of shell allows for designing a class of
ligands for functionalizing the nanoparticle5556
9
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of
ZnS B Quantum dots of different colors are presented with their corresponding core size
image of solution and photoluminescence spectra and color C Diagram representing the
quantum confinement and the change in band gap energy as material size decreases below
the Bohr-exciton radius Here CB and VB represent the conduction and valence band
respectively and Eg represent the band gap energies Image adapted with permission
Copyright 2011 American Chemical Society60
The resulting particles have been incorporated into biological systems using surface ligands
with chemistry that allows the crystals to be stable in aqueous and physiological buffers6162
Further functionalization of these ligands has also allowed for the integration of biomolecules like
nucleic acids63 and proteins64 and polymers like polyethylene glycol (PEG) allowing applications
that extend from biological imaging65 to diagnostic device development and commercial
technologies566667 Optically quantum dots (QD) have broad absorption wavelengths (from the
UV into the visible) narrow and symmetrical emission photoluminescence (PL) profiles (25 nm
of full width at half maximum) high quantum yields and photochemical stability59 These
spectral properties in addition to the large surface area of QDs make them favourable donors for
RET processes
10
131 Quantum Confinement and The Particle in a Box
A brief overview of the quantum mechanics related to QDs will be discussed before
detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids
As the semiconducting material that composes QDs transitions from the bulk scale to the nano-
scale the valence and conductance bands of the semiconductor material split into discrete
energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the
composite of materials however for nanomaterials the band gap can also be tuned by modulating
the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the
dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like
CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an
electron-hole pair When an electron is excited by a photon of greater energy than the band gap
(the probability increases at higher energies yielding broad absorption spectra) the separation of
the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used
to describe this phenomenon is called quantum confinement and the model that best describes it is
the particle in a box575960
In this model a particle is said to be confined in a symmetrical box (of diameter a) where
the center of the box is denoted as = 0 and the edges of the box are denoted as = (
( Here
the potential energy inside the box +( le le
(- is said to be zero and the potential energy outside
the box + le ( ge
(- is said to be infinite The resulting probability of finding a particle outside
the confines of the box is zero 0 = 0 + le ( ge
(-1 and the discrete energy
eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of
interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material
The surface of the material represents the impenetrable barrier (potential energy is infinity)
restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869
As size of the QDs decreases the energy required for excitation increases because the
exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral
properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a
RET based system because the surface area of QDs allows for loading of multiple biomolecules
Thus multiple pathways of RET can take place that can collectively improve energy transfer
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
5
Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and
Crick Arrows on the ribbons represent the directionality bias for the single strands and
dimensions for the polymer are presented with one turn of the helix every 34 nm the
distance between base pairs every 034 nm and the distance between the phosphate
backbone and the central axis every 1 nm B shows the hydrogen bonding taking place
between complementary pairs of nucleobases as proposed by Chargaff with adenine (A)
having two hydrogen bonds with thymine (T) and guanine (G) having three hydrogen bonds
with cytosine (C) Image was adapted with permission Copyright Nature Education 201331
122 Thermodynamics of DNA Hybridization
Design and development of DNA-based technologies have been guided by the
thermodynamic modelling of hybridization Techniques like PCR3233 and isothermal
amplification34 rely on accurate control over the annealing of primers and DNA sensors often uses
temperature and chaotropic agents for achieving selectivity35 One of the useful tools for modelling
hybridization and mismatch-based interactions is the nearest neighbor method (NN)36 To explore
the strategy between the NN method and hybridization of DNA it is useful to understand some
details related to predicting the melting temperature (Tm)
First the system of interest will be defined at the equilibrium of dsDNA and ssDNA at the
point where both populations are equal ie half the strands of DNA are in the double helix state
and the other half are single-stranded and are often in various conformations Tm is the temperature
6
at which this equilibrium is found Next the equilibrium constant Keq is defined as being a ratio
of the concentration of dsDNA and ssDNA (as stated in Equation 1) A second expression can be
derived from the Vanrsquot Hoff equation (as stated in Equation 2) where ∆Ho and ∆So are the standard
enthalpy and entropy of hybridization and lnCT is the natural logarithm of the total strand
concentration This second equation can be used to calculate the thermodynamic parameters
related to Tm with the same being true vice versa37
Equation 1 = [][]
Equation 2 = ∆∆
With this foundation investigation into the NN method for modelling can be undertaken
The thermodynamics associated with a base pair are related to some degree with neighboring base
pairs Free energy values and other related parameters have been determined experimentally for
model oligonucleotide sequences This information is then used in conjunction with the nearest
neighbor algorithm (as presented in Equation 3) for obtaining the Tm for the strand of interest
Here base pair doublets are considered for sequence stability with ten unique combinations of
doublets (5rsquo-3rsquo) CG GC AT AA (also = TT) AG (also = CT) AC (also = GT) GA (also = TC)
GG (also = CC) TG (also = CA)38
Equation 3 ∆ = ∆ + ∆ + sum ∆
Equation 4 ∆ = ∆ minus ∆
In Equation 3 the ∆Gi(total) refers to the free energy of the strand of interest ∆G(init)
refers to the free energy of the strand of initiating base pair ∆G(sym) refers to the free energy of
symmetry Gj refers to the free energy associated with one of ten nearest neighbor stacking
interactions and nij is the appearance rate of the stacking interaction of interest Thermodynamic
parameters are also present for entropy and enthalpy allowing for the calculation of the Tm using
Equation 43638 Higher Tm values indicate greater stability than lower Tm values39ndash42 The NN
method can also be used along with a database of mismatch energetics to determine the
thermodynamic parameters related to those sequences
7
Tm values when used in conjunction with the free energies provide a theoretical basis for
designing probe ndash capture strand interactions This understanding can be useful when designing
wash conditions that control stringency for oligonucleotides composed of sequences with high
similarity Stringency control can be achieved using higher temperature (because increasing
temperature results in de-annealing of sequences and has greater effect on hybrids with partial
complementarity)43 by controlling the ionic strength of hybridization44 and via chaotropic agents
such as formamide45 and urea46 Although all strategies are valid for reducing false positive signals
(that arise from partially complementary strands of oligonucleotides) the use of washes containing
chaotropic agents may be more applicable for the POC given that temperature control requires a
temperature module
Chaotropic agents like formamide lower the melting temperature of duplex DNA by
engaging with the hydrogen bond network of DNA The degree by which temperature is lowered
depends on the GC content the conformations of single and duplex forms and the hydration state
of the strand (but typically can range 24 ndash 29 degC per mole of formamide)45 Chemically
formamide strongly associates with DNA is capable of four hydrogen bonds (same as water) and
is a stronger hydrogen bond acceptor than water Formamide ndash water bonds have been reported to
be 20 stronger than water ndash water bonds and it is accepted that formamide engages the hydration
network around DNA4547 Thus using formamide in washes for DNA hybridization can lower the
melting temperature favoring fully complementary hybrids over partially complementary hybrids
123 Notes and Considerations for POC Application
Developing a DNA screening device for the POC application requires consideration of the
many challenges faced by clinicians When screening genetic samples from blood it is important
to note that samples are often complex with proteins and other type of biomolecules (in addition
to cellular debris) and these materials may occlude the signal generated from target detection48
Another challenge to note for nucleic acid-based diagnostics is the low amount of target present in
clinical samples For example one milliliter of human blood contains approximately 107
leukocytes corresponding to femtomolar quantities (fM or attomoles 10-18 moles) of target nucleic
acid Thus detection strategies requiring hybridization-based assay require enzymatic
amplification of the target materials or improved analytical figures of merit for application in
POC49 Presently there are many different strategies for enzymatic amplification of nucleic acids
8
including polymerase chain reaction32 loop-mediated isothermal amplification34 helicase-
dependent isothermal amplification50 and recombinase polymerase amplification51 Post
amplification targets are often detected using hybridization-based assays using Watson-Crick base
pairing for detection of targets of interest Typically capture probes of complementary sequence
to targets are immobilized on a surface and the presence of target forms hybrids that are transduced
via electrochemical5253 or optical strategies54 Nano-surfaces can be integrated into this detection
strategy with oligonucleotides immobilized on the surface of nanoparticles allowing for
transduction via near-field phenomenon
13 Quantum dots
Nanomaterials based on gold and semiconductor composites have had a significant impact
across many different research fields including the chemical physical and biological sciences
Interest in nanoparticles has been driven due to the unique fundamental properties of these
materials as they approach and occupy size regions between bulk material and isolated atoms
Luminescent semiconductor-based quantum dots (QDs) in particular have attracted attention due
to their unique electro-optical properties arising from small size scales (typically ranging from
2 ndash 10 nm and consisting of 102 ndash 104 atoms per crystal) The key factors of interest for these
particles are material composition and size with a combination of the two giving rise to control of
physical properties such as the spectral profile and photon band gap energies55ndash59
There are many strategies for preparing and tuning the electro-optical properties of QDs
but some of the most studied from a synthetic perspective are based on binary composites of
elements from groups II-VI (like CdSe CdS or CdTe) and III-V (like InAs)55ndash58 For binary
composites luminescent properties can be controlled by choice of materials (selecting specific
regions of the UV-vis spectrum) and by control of size whereby smaller crystals are blue-shifted
and larger crystals are red-shifted5759 QDs used in most biological investigations are constructed
in a coreshell manner where the core is composed on a composite of materials previously
mentioned and the shell is composed of an inert coating (ie ZnS see Figure 2A) The QD shell
protects the nanoparticle from environmental degradation forming a protective layer and provides
a larger potential energy barrier for confining the exciton The shell material also provides a
synthetic strategy for controlling the core size and the type of shell allows for designing a class of
ligands for functionalizing the nanoparticle5556
9
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of
ZnS B Quantum dots of different colors are presented with their corresponding core size
image of solution and photoluminescence spectra and color C Diagram representing the
quantum confinement and the change in band gap energy as material size decreases below
the Bohr-exciton radius Here CB and VB represent the conduction and valence band
respectively and Eg represent the band gap energies Image adapted with permission
Copyright 2011 American Chemical Society60
The resulting particles have been incorporated into biological systems using surface ligands
with chemistry that allows the crystals to be stable in aqueous and physiological buffers6162
Further functionalization of these ligands has also allowed for the integration of biomolecules like
nucleic acids63 and proteins64 and polymers like polyethylene glycol (PEG) allowing applications
that extend from biological imaging65 to diagnostic device development and commercial
technologies566667 Optically quantum dots (QD) have broad absorption wavelengths (from the
UV into the visible) narrow and symmetrical emission photoluminescence (PL) profiles (25 nm
of full width at half maximum) high quantum yields and photochemical stability59 These
spectral properties in addition to the large surface area of QDs make them favourable donors for
RET processes
10
131 Quantum Confinement and The Particle in a Box
A brief overview of the quantum mechanics related to QDs will be discussed before
detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids
As the semiconducting material that composes QDs transitions from the bulk scale to the nano-
scale the valence and conductance bands of the semiconductor material split into discrete
energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the
composite of materials however for nanomaterials the band gap can also be tuned by modulating
the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the
dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like
CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an
electron-hole pair When an electron is excited by a photon of greater energy than the band gap
(the probability increases at higher energies yielding broad absorption spectra) the separation of
the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used
to describe this phenomenon is called quantum confinement and the model that best describes it is
the particle in a box575960
In this model a particle is said to be confined in a symmetrical box (of diameter a) where
the center of the box is denoted as = 0 and the edges of the box are denoted as = (
( Here
the potential energy inside the box +( le le
(- is said to be zero and the potential energy outside
the box + le ( ge
(- is said to be infinite The resulting probability of finding a particle outside
the confines of the box is zero 0 = 0 + le ( ge
(-1 and the discrete energy
eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of
interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material
The surface of the material represents the impenetrable barrier (potential energy is infinity)
restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869
As size of the QDs decreases the energy required for excitation increases because the
exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral
properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a
RET based system because the surface area of QDs allows for loading of multiple biomolecules
Thus multiple pathways of RET can take place that can collectively improve energy transfer
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
6
at which this equilibrium is found Next the equilibrium constant Keq is defined as being a ratio
of the concentration of dsDNA and ssDNA (as stated in Equation 1) A second expression can be
derived from the Vanrsquot Hoff equation (as stated in Equation 2) where ∆Ho and ∆So are the standard
enthalpy and entropy of hybridization and lnCT is the natural logarithm of the total strand
concentration This second equation can be used to calculate the thermodynamic parameters
related to Tm with the same being true vice versa37
Equation 1 = [][]
Equation 2 = ∆∆
With this foundation investigation into the NN method for modelling can be undertaken
The thermodynamics associated with a base pair are related to some degree with neighboring base
pairs Free energy values and other related parameters have been determined experimentally for
model oligonucleotide sequences This information is then used in conjunction with the nearest
neighbor algorithm (as presented in Equation 3) for obtaining the Tm for the strand of interest
Here base pair doublets are considered for sequence stability with ten unique combinations of
doublets (5rsquo-3rsquo) CG GC AT AA (also = TT) AG (also = CT) AC (also = GT) GA (also = TC)
GG (also = CC) TG (also = CA)38
Equation 3 ∆ = ∆ + ∆ + sum ∆
Equation 4 ∆ = ∆ minus ∆
In Equation 3 the ∆Gi(total) refers to the free energy of the strand of interest ∆G(init)
refers to the free energy of the strand of initiating base pair ∆G(sym) refers to the free energy of
symmetry Gj refers to the free energy associated with one of ten nearest neighbor stacking
interactions and nij is the appearance rate of the stacking interaction of interest Thermodynamic
parameters are also present for entropy and enthalpy allowing for the calculation of the Tm using
Equation 43638 Higher Tm values indicate greater stability than lower Tm values39ndash42 The NN
method can also be used along with a database of mismatch energetics to determine the
thermodynamic parameters related to those sequences
7
Tm values when used in conjunction with the free energies provide a theoretical basis for
designing probe ndash capture strand interactions This understanding can be useful when designing
wash conditions that control stringency for oligonucleotides composed of sequences with high
similarity Stringency control can be achieved using higher temperature (because increasing
temperature results in de-annealing of sequences and has greater effect on hybrids with partial
complementarity)43 by controlling the ionic strength of hybridization44 and via chaotropic agents
such as formamide45 and urea46 Although all strategies are valid for reducing false positive signals
(that arise from partially complementary strands of oligonucleotides) the use of washes containing
chaotropic agents may be more applicable for the POC given that temperature control requires a
temperature module
Chaotropic agents like formamide lower the melting temperature of duplex DNA by
engaging with the hydrogen bond network of DNA The degree by which temperature is lowered
depends on the GC content the conformations of single and duplex forms and the hydration state
of the strand (but typically can range 24 ndash 29 degC per mole of formamide)45 Chemically
formamide strongly associates with DNA is capable of four hydrogen bonds (same as water) and
is a stronger hydrogen bond acceptor than water Formamide ndash water bonds have been reported to
be 20 stronger than water ndash water bonds and it is accepted that formamide engages the hydration
network around DNA4547 Thus using formamide in washes for DNA hybridization can lower the
melting temperature favoring fully complementary hybrids over partially complementary hybrids
123 Notes and Considerations for POC Application
Developing a DNA screening device for the POC application requires consideration of the
many challenges faced by clinicians When screening genetic samples from blood it is important
to note that samples are often complex with proteins and other type of biomolecules (in addition
to cellular debris) and these materials may occlude the signal generated from target detection48
Another challenge to note for nucleic acid-based diagnostics is the low amount of target present in
clinical samples For example one milliliter of human blood contains approximately 107
leukocytes corresponding to femtomolar quantities (fM or attomoles 10-18 moles) of target nucleic
acid Thus detection strategies requiring hybridization-based assay require enzymatic
amplification of the target materials or improved analytical figures of merit for application in
POC49 Presently there are many different strategies for enzymatic amplification of nucleic acids
8
including polymerase chain reaction32 loop-mediated isothermal amplification34 helicase-
dependent isothermal amplification50 and recombinase polymerase amplification51 Post
amplification targets are often detected using hybridization-based assays using Watson-Crick base
pairing for detection of targets of interest Typically capture probes of complementary sequence
to targets are immobilized on a surface and the presence of target forms hybrids that are transduced
via electrochemical5253 or optical strategies54 Nano-surfaces can be integrated into this detection
strategy with oligonucleotides immobilized on the surface of nanoparticles allowing for
transduction via near-field phenomenon
13 Quantum dots
Nanomaterials based on gold and semiconductor composites have had a significant impact
across many different research fields including the chemical physical and biological sciences
Interest in nanoparticles has been driven due to the unique fundamental properties of these
materials as they approach and occupy size regions between bulk material and isolated atoms
Luminescent semiconductor-based quantum dots (QDs) in particular have attracted attention due
to their unique electro-optical properties arising from small size scales (typically ranging from
2 ndash 10 nm and consisting of 102 ndash 104 atoms per crystal) The key factors of interest for these
particles are material composition and size with a combination of the two giving rise to control of
physical properties such as the spectral profile and photon band gap energies55ndash59
There are many strategies for preparing and tuning the electro-optical properties of QDs
but some of the most studied from a synthetic perspective are based on binary composites of
elements from groups II-VI (like CdSe CdS or CdTe) and III-V (like InAs)55ndash58 For binary
composites luminescent properties can be controlled by choice of materials (selecting specific
regions of the UV-vis spectrum) and by control of size whereby smaller crystals are blue-shifted
and larger crystals are red-shifted5759 QDs used in most biological investigations are constructed
in a coreshell manner where the core is composed on a composite of materials previously
mentioned and the shell is composed of an inert coating (ie ZnS see Figure 2A) The QD shell
protects the nanoparticle from environmental degradation forming a protective layer and provides
a larger potential energy barrier for confining the exciton The shell material also provides a
synthetic strategy for controlling the core size and the type of shell allows for designing a class of
ligands for functionalizing the nanoparticle5556
9
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of
ZnS B Quantum dots of different colors are presented with their corresponding core size
image of solution and photoluminescence spectra and color C Diagram representing the
quantum confinement and the change in band gap energy as material size decreases below
the Bohr-exciton radius Here CB and VB represent the conduction and valence band
respectively and Eg represent the band gap energies Image adapted with permission
Copyright 2011 American Chemical Society60
The resulting particles have been incorporated into biological systems using surface ligands
with chemistry that allows the crystals to be stable in aqueous and physiological buffers6162
Further functionalization of these ligands has also allowed for the integration of biomolecules like
nucleic acids63 and proteins64 and polymers like polyethylene glycol (PEG) allowing applications
that extend from biological imaging65 to diagnostic device development and commercial
technologies566667 Optically quantum dots (QD) have broad absorption wavelengths (from the
UV into the visible) narrow and symmetrical emission photoluminescence (PL) profiles (25 nm
of full width at half maximum) high quantum yields and photochemical stability59 These
spectral properties in addition to the large surface area of QDs make them favourable donors for
RET processes
10
131 Quantum Confinement and The Particle in a Box
A brief overview of the quantum mechanics related to QDs will be discussed before
detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids
As the semiconducting material that composes QDs transitions from the bulk scale to the nano-
scale the valence and conductance bands of the semiconductor material split into discrete
energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the
composite of materials however for nanomaterials the band gap can also be tuned by modulating
the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the
dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like
CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an
electron-hole pair When an electron is excited by a photon of greater energy than the band gap
(the probability increases at higher energies yielding broad absorption spectra) the separation of
the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used
to describe this phenomenon is called quantum confinement and the model that best describes it is
the particle in a box575960
In this model a particle is said to be confined in a symmetrical box (of diameter a) where
the center of the box is denoted as = 0 and the edges of the box are denoted as = (
( Here
the potential energy inside the box +( le le
(- is said to be zero and the potential energy outside
the box + le ( ge
(- is said to be infinite The resulting probability of finding a particle outside
the confines of the box is zero 0 = 0 + le ( ge
(-1 and the discrete energy
eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of
interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material
The surface of the material represents the impenetrable barrier (potential energy is infinity)
restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869
As size of the QDs decreases the energy required for excitation increases because the
exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral
properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a
RET based system because the surface area of QDs allows for loading of multiple biomolecules
Thus multiple pathways of RET can take place that can collectively improve energy transfer
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
7
Tm values when used in conjunction with the free energies provide a theoretical basis for
designing probe ndash capture strand interactions This understanding can be useful when designing
wash conditions that control stringency for oligonucleotides composed of sequences with high
similarity Stringency control can be achieved using higher temperature (because increasing
temperature results in de-annealing of sequences and has greater effect on hybrids with partial
complementarity)43 by controlling the ionic strength of hybridization44 and via chaotropic agents
such as formamide45 and urea46 Although all strategies are valid for reducing false positive signals
(that arise from partially complementary strands of oligonucleotides) the use of washes containing
chaotropic agents may be more applicable for the POC given that temperature control requires a
temperature module
Chaotropic agents like formamide lower the melting temperature of duplex DNA by
engaging with the hydrogen bond network of DNA The degree by which temperature is lowered
depends on the GC content the conformations of single and duplex forms and the hydration state
of the strand (but typically can range 24 ndash 29 degC per mole of formamide)45 Chemically
formamide strongly associates with DNA is capable of four hydrogen bonds (same as water) and
is a stronger hydrogen bond acceptor than water Formamide ndash water bonds have been reported to
be 20 stronger than water ndash water bonds and it is accepted that formamide engages the hydration
network around DNA4547 Thus using formamide in washes for DNA hybridization can lower the
melting temperature favoring fully complementary hybrids over partially complementary hybrids
123 Notes and Considerations for POC Application
Developing a DNA screening device for the POC application requires consideration of the
many challenges faced by clinicians When screening genetic samples from blood it is important
to note that samples are often complex with proteins and other type of biomolecules (in addition
to cellular debris) and these materials may occlude the signal generated from target detection48
Another challenge to note for nucleic acid-based diagnostics is the low amount of target present in
clinical samples For example one milliliter of human blood contains approximately 107
leukocytes corresponding to femtomolar quantities (fM or attomoles 10-18 moles) of target nucleic
acid Thus detection strategies requiring hybridization-based assay require enzymatic
amplification of the target materials or improved analytical figures of merit for application in
POC49 Presently there are many different strategies for enzymatic amplification of nucleic acids
8
including polymerase chain reaction32 loop-mediated isothermal amplification34 helicase-
dependent isothermal amplification50 and recombinase polymerase amplification51 Post
amplification targets are often detected using hybridization-based assays using Watson-Crick base
pairing for detection of targets of interest Typically capture probes of complementary sequence
to targets are immobilized on a surface and the presence of target forms hybrids that are transduced
via electrochemical5253 or optical strategies54 Nano-surfaces can be integrated into this detection
strategy with oligonucleotides immobilized on the surface of nanoparticles allowing for
transduction via near-field phenomenon
13 Quantum dots
Nanomaterials based on gold and semiconductor composites have had a significant impact
across many different research fields including the chemical physical and biological sciences
Interest in nanoparticles has been driven due to the unique fundamental properties of these
materials as they approach and occupy size regions between bulk material and isolated atoms
Luminescent semiconductor-based quantum dots (QDs) in particular have attracted attention due
to their unique electro-optical properties arising from small size scales (typically ranging from
2 ndash 10 nm and consisting of 102 ndash 104 atoms per crystal) The key factors of interest for these
particles are material composition and size with a combination of the two giving rise to control of
physical properties such as the spectral profile and photon band gap energies55ndash59
There are many strategies for preparing and tuning the electro-optical properties of QDs
but some of the most studied from a synthetic perspective are based on binary composites of
elements from groups II-VI (like CdSe CdS or CdTe) and III-V (like InAs)55ndash58 For binary
composites luminescent properties can be controlled by choice of materials (selecting specific
regions of the UV-vis spectrum) and by control of size whereby smaller crystals are blue-shifted
and larger crystals are red-shifted5759 QDs used in most biological investigations are constructed
in a coreshell manner where the core is composed on a composite of materials previously
mentioned and the shell is composed of an inert coating (ie ZnS see Figure 2A) The QD shell
protects the nanoparticle from environmental degradation forming a protective layer and provides
a larger potential energy barrier for confining the exciton The shell material also provides a
synthetic strategy for controlling the core size and the type of shell allows for designing a class of
ligands for functionalizing the nanoparticle5556
9
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of
ZnS B Quantum dots of different colors are presented with their corresponding core size
image of solution and photoluminescence spectra and color C Diagram representing the
quantum confinement and the change in band gap energy as material size decreases below
the Bohr-exciton radius Here CB and VB represent the conduction and valence band
respectively and Eg represent the band gap energies Image adapted with permission
Copyright 2011 American Chemical Society60
The resulting particles have been incorporated into biological systems using surface ligands
with chemistry that allows the crystals to be stable in aqueous and physiological buffers6162
Further functionalization of these ligands has also allowed for the integration of biomolecules like
nucleic acids63 and proteins64 and polymers like polyethylene glycol (PEG) allowing applications
that extend from biological imaging65 to diagnostic device development and commercial
technologies566667 Optically quantum dots (QD) have broad absorption wavelengths (from the
UV into the visible) narrow and symmetrical emission photoluminescence (PL) profiles (25 nm
of full width at half maximum) high quantum yields and photochemical stability59 These
spectral properties in addition to the large surface area of QDs make them favourable donors for
RET processes
10
131 Quantum Confinement and The Particle in a Box
A brief overview of the quantum mechanics related to QDs will be discussed before
detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids
As the semiconducting material that composes QDs transitions from the bulk scale to the nano-
scale the valence and conductance bands of the semiconductor material split into discrete
energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the
composite of materials however for nanomaterials the band gap can also be tuned by modulating
the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the
dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like
CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an
electron-hole pair When an electron is excited by a photon of greater energy than the band gap
(the probability increases at higher energies yielding broad absorption spectra) the separation of
the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used
to describe this phenomenon is called quantum confinement and the model that best describes it is
the particle in a box575960
In this model a particle is said to be confined in a symmetrical box (of diameter a) where
the center of the box is denoted as = 0 and the edges of the box are denoted as = (
( Here
the potential energy inside the box +( le le
(- is said to be zero and the potential energy outside
the box + le ( ge
(- is said to be infinite The resulting probability of finding a particle outside
the confines of the box is zero 0 = 0 + le ( ge
(-1 and the discrete energy
eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of
interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material
The surface of the material represents the impenetrable barrier (potential energy is infinity)
restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869
As size of the QDs decreases the energy required for excitation increases because the
exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral
properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a
RET based system because the surface area of QDs allows for loading of multiple biomolecules
Thus multiple pathways of RET can take place that can collectively improve energy transfer
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
8
including polymerase chain reaction32 loop-mediated isothermal amplification34 helicase-
dependent isothermal amplification50 and recombinase polymerase amplification51 Post
amplification targets are often detected using hybridization-based assays using Watson-Crick base
pairing for detection of targets of interest Typically capture probes of complementary sequence
to targets are immobilized on a surface and the presence of target forms hybrids that are transduced
via electrochemical5253 or optical strategies54 Nano-surfaces can be integrated into this detection
strategy with oligonucleotides immobilized on the surface of nanoparticles allowing for
transduction via near-field phenomenon
13 Quantum dots
Nanomaterials based on gold and semiconductor composites have had a significant impact
across many different research fields including the chemical physical and biological sciences
Interest in nanoparticles has been driven due to the unique fundamental properties of these
materials as they approach and occupy size regions between bulk material and isolated atoms
Luminescent semiconductor-based quantum dots (QDs) in particular have attracted attention due
to their unique electro-optical properties arising from small size scales (typically ranging from
2 ndash 10 nm and consisting of 102 ndash 104 atoms per crystal) The key factors of interest for these
particles are material composition and size with a combination of the two giving rise to control of
physical properties such as the spectral profile and photon band gap energies55ndash59
There are many strategies for preparing and tuning the electro-optical properties of QDs
but some of the most studied from a synthetic perspective are based on binary composites of
elements from groups II-VI (like CdSe CdS or CdTe) and III-V (like InAs)55ndash58 For binary
composites luminescent properties can be controlled by choice of materials (selecting specific
regions of the UV-vis spectrum) and by control of size whereby smaller crystals are blue-shifted
and larger crystals are red-shifted5759 QDs used in most biological investigations are constructed
in a coreshell manner where the core is composed on a composite of materials previously
mentioned and the shell is composed of an inert coating (ie ZnS see Figure 2A) The QD shell
protects the nanoparticle from environmental degradation forming a protective layer and provides
a larger potential energy barrier for confining the exciton The shell material also provides a
synthetic strategy for controlling the core size and the type of shell allows for designing a class of
ligands for functionalizing the nanoparticle5556
9
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of
ZnS B Quantum dots of different colors are presented with their corresponding core size
image of solution and photoluminescence spectra and color C Diagram representing the
quantum confinement and the change in band gap energy as material size decreases below
the Bohr-exciton radius Here CB and VB represent the conduction and valence band
respectively and Eg represent the band gap energies Image adapted with permission
Copyright 2011 American Chemical Society60
The resulting particles have been incorporated into biological systems using surface ligands
with chemistry that allows the crystals to be stable in aqueous and physiological buffers6162
Further functionalization of these ligands has also allowed for the integration of biomolecules like
nucleic acids63 and proteins64 and polymers like polyethylene glycol (PEG) allowing applications
that extend from biological imaging65 to diagnostic device development and commercial
technologies566667 Optically quantum dots (QD) have broad absorption wavelengths (from the
UV into the visible) narrow and symmetrical emission photoluminescence (PL) profiles (25 nm
of full width at half maximum) high quantum yields and photochemical stability59 These
spectral properties in addition to the large surface area of QDs make them favourable donors for
RET processes
10
131 Quantum Confinement and The Particle in a Box
A brief overview of the quantum mechanics related to QDs will be discussed before
detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids
As the semiconducting material that composes QDs transitions from the bulk scale to the nano-
scale the valence and conductance bands of the semiconductor material split into discrete
energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the
composite of materials however for nanomaterials the band gap can also be tuned by modulating
the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the
dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like
CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an
electron-hole pair When an electron is excited by a photon of greater energy than the band gap
(the probability increases at higher energies yielding broad absorption spectra) the separation of
the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used
to describe this phenomenon is called quantum confinement and the model that best describes it is
the particle in a box575960
In this model a particle is said to be confined in a symmetrical box (of diameter a) where
the center of the box is denoted as = 0 and the edges of the box are denoted as = (
( Here
the potential energy inside the box +( le le
(- is said to be zero and the potential energy outside
the box + le ( ge
(- is said to be infinite The resulting probability of finding a particle outside
the confines of the box is zero 0 = 0 + le ( ge
(-1 and the discrete energy
eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of
interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material
The surface of the material represents the impenetrable barrier (potential energy is infinity)
restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869
As size of the QDs decreases the energy required for excitation increases because the
exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral
properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a
RET based system because the surface area of QDs allows for loading of multiple biomolecules
Thus multiple pathways of RET can take place that can collectively improve energy transfer
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
9
Figure 2A Representation of the core-shell model of quantum dots with corresponding high-
resolution TEM image Here core material is composed of CdSe and shell is composed of
ZnS B Quantum dots of different colors are presented with their corresponding core size
image of solution and photoluminescence spectra and color C Diagram representing the
quantum confinement and the change in band gap energy as material size decreases below
the Bohr-exciton radius Here CB and VB represent the conduction and valence band
respectively and Eg represent the band gap energies Image adapted with permission
Copyright 2011 American Chemical Society60
The resulting particles have been incorporated into biological systems using surface ligands
with chemistry that allows the crystals to be stable in aqueous and physiological buffers6162
Further functionalization of these ligands has also allowed for the integration of biomolecules like
nucleic acids63 and proteins64 and polymers like polyethylene glycol (PEG) allowing applications
that extend from biological imaging65 to diagnostic device development and commercial
technologies566667 Optically quantum dots (QD) have broad absorption wavelengths (from the
UV into the visible) narrow and symmetrical emission photoluminescence (PL) profiles (25 nm
of full width at half maximum) high quantum yields and photochemical stability59 These
spectral properties in addition to the large surface area of QDs make them favourable donors for
RET processes
10
131 Quantum Confinement and The Particle in a Box
A brief overview of the quantum mechanics related to QDs will be discussed before
detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids
As the semiconducting material that composes QDs transitions from the bulk scale to the nano-
scale the valence and conductance bands of the semiconductor material split into discrete
energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the
composite of materials however for nanomaterials the band gap can also be tuned by modulating
the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the
dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like
CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an
electron-hole pair When an electron is excited by a photon of greater energy than the band gap
(the probability increases at higher energies yielding broad absorption spectra) the separation of
the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used
to describe this phenomenon is called quantum confinement and the model that best describes it is
the particle in a box575960
In this model a particle is said to be confined in a symmetrical box (of diameter a) where
the center of the box is denoted as = 0 and the edges of the box are denoted as = (
( Here
the potential energy inside the box +( le le
(- is said to be zero and the potential energy outside
the box + le ( ge
(- is said to be infinite The resulting probability of finding a particle outside
the confines of the box is zero 0 = 0 + le ( ge
(-1 and the discrete energy
eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of
interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material
The surface of the material represents the impenetrable barrier (potential energy is infinity)
restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869
As size of the QDs decreases the energy required for excitation increases because the
exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral
properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a
RET based system because the surface area of QDs allows for loading of multiple biomolecules
Thus multiple pathways of RET can take place that can collectively improve energy transfer
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
10
131 Quantum Confinement and The Particle in a Box
A brief overview of the quantum mechanics related to QDs will be discussed before
detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids
As the semiconducting material that composes QDs transitions from the bulk scale to the nano-
scale the valence and conductance bands of the semiconductor material split into discrete
energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the
composite of materials however for nanomaterials the band gap can also be tuned by modulating
the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the
dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like
CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an
electron-hole pair When an electron is excited by a photon of greater energy than the band gap
(the probability increases at higher energies yielding broad absorption spectra) the separation of
the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used
to describe this phenomenon is called quantum confinement and the model that best describes it is
the particle in a box575960
In this model a particle is said to be confined in a symmetrical box (of diameter a) where
the center of the box is denoted as = 0 and the edges of the box are denoted as = (
( Here
the potential energy inside the box +( le le
(- is said to be zero and the potential energy outside
the box + le ( ge
(- is said to be infinite The resulting probability of finding a particle outside
the confines of the box is zero 0 = 0 + le ( ge
(-1 and the discrete energy
eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of
interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material
The surface of the material represents the impenetrable barrier (potential energy is infinity)
restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869
As size of the QDs decreases the energy required for excitation increases because the
exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral
properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a
RET based system because the surface area of QDs allows for loading of multiple biomolecules
Thus multiple pathways of RET can take place that can collectively improve energy transfer
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
11
efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric
data processing approach where acceptor and QD donor emission are tracked together thus greater
precision for biological interactions is achieved70
14 Fluorescence and Resonance Energy Transfer
The ideas related to fluorescence are important for building an understanding of the details
related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos
Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71
The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz
and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73
for more details on theory of FRET
141 Fluorescence Resonance Energy Transfer (FRET)
Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance
energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)
undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)
The result of this distance-dependent interaction forms the basis of bio-recognition based assays73
Although the theory of FRET has been discussed in detail elsewhere7273 the specific application
of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that
make them excellent donors in FRET and two strong arguments for their advantage in FRET assays
involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster
distance (see Equation 6)7073
Equation 5 = = sum gt frasl ABsum gt frasl A
asymp gtAAgtA
Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU
S TUU
The efficiency of FRET details the degree to which energy transfer between the donor and
the acceptor is achieved This is primarily a function of the number of acceptors and the distances
related to the FRET pair For an individual QD of (near) spherical structure multiple FRET
acceptors are predicted to self-assemble on the surface of the crystal The specific location and
orientation of the acceptors are predicted to vary However the variations can be assumed to be
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
12
averaged In solution these acceptors are expected to self-assemble in all directions and the
resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From
Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors
results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as
described by Equation 5 When QDs are immobilized on a surface the number of acceptors
coordinating on the nanoparticle are expected to be less than in solution because a portion of the
QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean
that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can
undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met
Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET
is represented by E the Foumlrster distance is represented by R0 the distance between the donor and
the acceptor is represented by r and the total number of acceptors is represented by a7073
Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of
colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)
Change in FRET efficiency based on changes in the distance between donor and acceptor
(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by
interacting with adjacent acceptors Image acquired with permission from Algar et al70
Copyright Elsevier 2010
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
13
The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and
represents the distance at which the efficiency of energy transfer is at 50 Parameters from both
the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor
is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral
overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the
molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be
achieved with QDs because their spectral properties as FRET donors can be controlled affording
large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow
and the photoluminescence (PL) wavelength range is tunable based on control of the size of the
nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD
emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash
09) with absorption profiles extending from the emission region to high energy UV Thus QDs
can be excited at higher energies avoiding excitation of the acceptor from QD light sources In
addition to excitation wavelength the excitation power required for QDs is lower than molecular
dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower
intensity excitation minimizes the rate of photobleaching These properties make QDs good donors
in FRET based processes and biosensors that integrate QD based FRET for sensing
biomolecules6070
Fluorescence is a high-sensitivity method among oligonucleotide-based detection
strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via
the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some
applications74 The performance of fluorescence-based systems can be further improved by
integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer
(FRET) strategy for application in microPADs75 A representation of two analysis formats based on
labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the
methodology proposed in the work herein
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
14
Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in
blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)
functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3
on the proximal end and upon hybridization is brought to proximity with gQDs allowing for
FRET to take place (B) In sandwich assay format the probe strand hybridizes with the
target strand (seen in red) such that there is an overhang on the distal end Reporter strand
(seen in green) hybridizes with the overhang region of the target strand bringing to proximity
the Cy3 label on the proximal end of the reporter
15 Paper Based Analytical Devices
Advances in bioassays and sensing technologies for point-of-care (POC) or resource-
limited settings have been guided by recommendations of the World Health Organizationrsquos
ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid
and robust equipment free and deliverable to those who need them1976 Paper as a substrate has
been growing in popularity for device development primarily due to this criteria for POC devices
Paper based analytical devices (PADs) are affordable to manufacture with commercial options
offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-
based technology77 and easy transport is possible via stacking sheets of devices19 The wicking
properties of paper allow for elimination of pumps and power supply modules often required for
microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
15
modification of cellulose for developing sensing technology PADs can also be incinerated after
use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a
multitude of advantages PADs were chosen as a platform for developing sensing chemistry and
the following sections will introduce cellulose modification and fluorescence transduction
strategies used in conjunction with paper
151 Paper Substrates for Sensing Technology Overview
Paper is a suitable substrate for development of analytical devices with fluidic capabilities
(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been
implemented as a platform for screening and semi-quantitative assays of biomarkers offering
reliable performance at low cost with ease of use and disposal79 As an emerging technology for
POC application microPADs are uniquely poised to function as systems that can process raw samples
and then complete an analysis to yield information regarding the genetic basis of disease80
Research within the microPAD field has often focused on individual functional components of a
complete device including sample preparation81 (ie extraction of analytes from complex
samples) amplification of analytes of interest82ndash84 and detection commonly using
electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-
field applications the preference is isothermal enzymatic amplification yielding products that are
either labelled or unlabelled with dyes depending on the detection scheme and the desired
analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification
can be achieved on paper substrates91 providing product for the transduction step which is the
focus of the work in this investigation
152 Cellulose Modification and Smartphone-based Detection
Whatman chromatography paper is one of the most common substrates for developing
PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose
chemistry is well defined but only specific modifications that do not alter the spectroscopic quality
of paper are suitable for PAD development Incompatible chemistry may discolour the paper and
this would create challenges for reproducibility and accuracy of sensing One of the strategies for
modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar
groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for
bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
16
amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic
acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to
nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers
was incorporated into this investigation due to the reproducibility and yield of the modification
reaction
Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde
functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society
of Chemistry 2016
Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)
fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and
is difficult to integrate into a portable system To overcome these challenges the camera (imaging)
technology in smartphones and personal electronic devices offer an effective compromise that is
readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with
microscopes but these portable digital cameras have advanced processing systems and computing
power in these devices that rival most personal computers Integration of smartphone technology
for colourimetric and fluorescence-based assays has been demonstrated for many applications
providing figures of merit that are comparable to most other commercially available imaging
technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye
labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a
limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In
contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-
based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus
a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR
gene on paper
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
17
16 Thesis Objectives and Contributions
Investigations of the detection of oligonucleotides in a paper matrix have primarily focused
on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101
The results of these investigations suggest potential for distinction between mismatches and this
has been examined using a paper-based format to detect a three-base pair deletion associated with
CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform
for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors
Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair
Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET
donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair
Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and
mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with
the intention of comparison of analytical performance to guide the subsequent development of an
amplification format in the future Smartphone imaging was used to collect PL data A schematic
detailing the operation of the paper-based assay is presented as Figure 6
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
18
Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)
Reaction zones consisted of chemically modified paper that were conjugated with gQD-
oligonucleotide probes Zones contained WT and MT controls and test zones where
unknown samples were spotted and imaged Detection was based on the principle of RET
with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)
Imaging used a smartphone camera with data processing by ImageJ to split the image to
RGB color channels
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
19
Chapter 2
Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera
Author Contribution Statement
All experimental work was done by K Malhotra All authors contributed to the
experimental design data analysis and preparation of the manuscript This chapter is based on the
following manuscript
Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane
Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization
Assay and a Smartphone Camera Manuscript submitted
21 Experimental
Reagents and Oligonucleotides
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak
photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from
Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade
1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-
glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F
ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous
ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-
aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-
Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system
(Milli-Q 18 M`cm-1) and were autoclaved prior to use
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
20
The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT
Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)
The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC
Table 2 Oligonucleotide Sequences used in Hybridization Assays
Name Sequence
CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo
CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo
CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo
CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo
CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo
CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo
CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG
TAG
CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo
TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =
Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter
211 Methods
2111 Preparation of QD-Probe Oligonucleotide Conjugates
In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL
at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione
(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified
CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and
oligonucleotide conjugated QDs is presented as follows
Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)
capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with
glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of
tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was
added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The
resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness
at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous
solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
21
100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a
vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous
(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was
centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant
was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer
precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL
of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using
UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102
GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)
oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single
or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ
reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times
molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM
borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an
orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo
The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours
to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an
orbital shaker The solution containing QD-probe conjugates was used without further purification
(unless otherwise stated) and stored at 4 degC98
2112 Solution-Phase Hybridization Assays
Solution-phase hybridization assays were conducted in triplicate and direct assay format
For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide
targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)
in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of
15 minutes prior to sample measurements
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
22
2113 Surface Modification of Paper with Imidazole Groups
Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper
substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN
solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde
functionalities that were further reacted via reductive amination to obtain imidazole groups on the
paper A detailed protocol for preparing paper substrates is presented as follows
Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose
chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software
The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8
format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm
Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were
printed with one pass at the high resolution using black wax (product number = 108R00930
although other wax colors could theoretically be used for printing without any impact on the
chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven
at 120 degC for 2 minutes
Modification of paper was based on a two-step reaction First cellulose was oxidized to
yield aldehyde groups and then an imidazole functionality was added via reductive amination87
Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In
a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water
and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then
placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which
the papers were washed Washing was accomplished by placing the papers in Milli-Q water and
agitating for 2 minutes after which the papers were dried in a desiccator overnight
Imidazole functionality was added to the aldehyde modified paper via reductive amination
with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160
mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were
spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an
hour
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
23
21131 Note on Troubleshooting Leaking of Paper Zones
A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the
paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC
In addition to this previous protocols for paper modification have reported the use of a 10 min
wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with
imidazole solution This step was modified to a BB wash for 10 min because it is believed that
addition of SDS was resulting in erosion of wax from paper substrates
Figure 7 Image of buffer solution leakage from hydrophilic paper zones
2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays
Hybridization assays on paper substrates were conducted using two formats direct assay and
sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on
imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH
925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR
MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing
with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a
smartphone camera Depending on the desired investigation (ie wash conditions for stringency)
a further wash step was done followed by drying under vacuum for an hour before imaging with a
smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper
zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide
targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room
temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
24
temperature before being washed with BBS for 30 sec Papers were then dried for an hour under
vacuum before imaging with a smartphone Depending on the desired investigation (ie wash
conditions for stringency) a further wash step was done followed by drying under vacuum for an
hour before imaging with a smartphone camera
212 Instrumentation
2121 PL Spectra and Digital Image Acquisition
PL spectra for hybridization assays done in solution-phase were acquired using a
QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The
excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive
R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL
spectra were calculated using Equation 7
Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt
1
Digital color images for paper substrates were acquired using an iPhone SE with the built-
in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)
was placed in front of the camera to prevent saturation of the detector and the imaging was done
in a dark room Default settings were used for all images with no alterations to exposure time or
detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science
Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to
illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured
using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power
meter model 1918-C Irvine California U S A) The measured power from the UV lamp was
44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05
cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios
(ratiometric response) from the digital images were calculated using Equation 8
Equation 8 bc = + =e=e
-
minus + =e=e
-
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
25
2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization
Data for a ratiometric format of signal transduction requires simultaneous measurement of
intensity from two wavelength bands associated with the PL of the RET donor and acceptor
Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor
PL associated with the green color channel and acceptor PL was associated with the red color
channel and dividing the average signal intensity of the red color channel with the green color
channel Images were processed using ImageJ software (version 149v National Institutes of
Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels
in the reaction zones on the paper substrates with the average signal obtained via measurement of
n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were
used as the brightest spots and served as background control Imaging was conducted in a dark
room using dried paper which has previously been reported to offer greater fluorescence
intensity98
22 Results and Discussion
221 FRET Pair Characterization (gQD ndash Cy3)
The optical signal from the bioassay explored in this investigation was based on the near-
field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism
was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm
Detection of target sequences of interest was observed as a decrease in the PL of the RET donor
and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target
the fluorescence from the paper zones were observed to change from green to yellow indicating
that RET was occurring (see Figure 8)
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
26
Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The
spectral overlap is represented by the shaded region Absorption is shown as dashed lines
and emission is shown as solid lines
Solution based measurements were done to determine the Foumlrster distance (Ro) using
where 9 refers to the refractive index of the surrounding medium (in this investigation a value of
133 was used) W( refers to the orientation factor (in this investigation a random orientation was
assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified
green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral
overlap interval (Z) was determined using
Equation 9 A = K PD Q BgtHK Q NGHgJ
In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_
is the molar extinction coefficient associated with the FRET acceptor as a function of ]
Equation 10 J = S TUVUUNUS TUU
222 Oligonucleotide Hybridization in Solution
Solution-phase assays were conducted to characterize the interaction between probe and
target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via
spectral analysis to obtain a ratiometric value for the interaction Normalized and background
corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to
the energy transfer process Background correction used the Cy3 dye emission spectra
corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra
corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD
0
05
1
15
2
25
3
400 450 500 550 600 650 700
No
rma
lize
d A
BS
PL
Sp
ect
ra
Wavelength (nm)
gQD ABS
Cy3 ABS
gQD EM
Cy3 EM
gQD Cy3
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
27
emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-
acceptor) and the background donor emission was subtracted (ie subscript D for donor) The
ratios obtained from this processing were further averaged using three measurements in total
A range of stoichiometric concentrations for gQDs-probe conjugates and targets were
investigated to obtain concentration-response curves for the different gQD-probe conjugates In
total two different types of conjugates were investigated in solution including gQD-WT probe
conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The
response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each
of the conjugates hybridization of two different types of targets were investigated Data points
shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to
CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR
WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT
Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids
Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET
signals from samples of FC hybrids vs PC hybrids)
Figure 9 Hybridization of the gQD-probe strands was investigated in solution by
fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)
CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT
target strands The concentration-response curves for the different gQD-probe conjugates
are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target
strands are seen in orange Normalized PL spectra for the calibration curves are shown for
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
28
B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (
indicates increasing target concentration)
It was found that the fully complementary (FC) hybrids were more stable
thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe
conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC
hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity
of the FC hybrids This data led us to believe that with wash stringency control sufficient
discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-
substrates may be accomplished
Figure 10 Representations of the two different direct assay formats investigated in solution
phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA
MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR
MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which
resulted in FRET
223 Oligonucleotide Hybridization in Paper Substrates
Selectivity of base pair hybridization of DNA strands can be controlled by environmental
manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted
by control of the ionic strength the pH of the hybridization solution and by altering the
thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide
Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide
stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the
hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
29
temperature depression caused by addition of formamide is dependent on factors including GC
composition of the oligonucleotide strand the helical conformation and the state of hydration
Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be
lower than those containing GC perhaps due to the different hydration pattern of AT containing
oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be
achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that
the paper undergoes the wash A preliminary investigation of the thermodynamic parameters
associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method
was used to determine the thermodynamic parameters associated with the expected probe ndash target
hybrids used in the design of this experiment42 The resulting data was used to interpret the
information produced from the FRET-based system undergoing wash conditions of various
stringencies
Investigation of the fluorescence response caused by hybridization within paper substrates
was accomplished by image analysis to obtain a ratiometric value for the FRET process
Background correction was accomplished using Equation 8 where the intensity of signal in the
paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity
of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of
the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor
(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript
D for donor) for each sample spot The data was further processed by obtaining an average value
of four background corrected paper zones for each sample concentration (example of images used
for data processing provided as Figure 11)
Figure 11 Digital smartphone image and the accompanying post-processing PL images (post
processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe
conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
30
Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol
(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of
spots that may not be visible otherwise
2231 Direct Assay Format
The direct assay made use of hybridization of probe strands with fluorescently labelled targets
Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or
gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands
CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different
variations of probe and target oligonucleotide conjugates were investigated as presented in Figure
12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31
kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and
(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for
WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in
stabilities indicate that careful control of formamide concentration may be sufficient to distinguish
between WT and MT gene fragments at room temperature
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
D MT Probe ndash WT Target
(8 Complementary Base Pairs with Probe)
∆Gmax = -14 kcal mol-1 ∆Gmax = -14 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 20 degC (293 K)
Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers
to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash
gQD gQD
gQD gQD
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
31
MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
2232 Sandwich Assay Format
A sandwich assay strategy was based on the step-wise hybridization of probe strands with
unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence
Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe
systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT
TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe
and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast
to direct assay the sandwich assay consists of two hybridization events Of the two hybridization
events only the first event was expected to yield partially complementary (PC) structures while
the second event was expected to always yield fully complementary (FC) structures For the first
hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-
1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are
PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe
ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with
those determined for the direct assay and as expected were higher than the values for hybrids (C)
and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich
assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a
PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates
(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted
to require wash conditions of greater stringency than other PC conjugates For the second
hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC
(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second
hybridization event in FC conjugates The result was that wash conditions required to achieve the
mismatch discrimination would also result in signal loss for FC conjugates because for a single
paper system FC hybrids were washed in the same conditions as PC hybrids
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
32
A WT Probe ndash WT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
B MT Probe ndash MT Target
(18 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1
Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)
C WT Probe ndash MT Target
(8 Complementary Base Pairs with Probe)
(FC with REP)
D MT Probe ndash WT Target
(14 Complementary Base Pairs with Probe)
(FC with REP)
∆Gmax = -14 kcal mol-1 ∆Gmax = -20 kcal mol-1
Theoretical Tm = 22 degC (295 K) Theoretical Tm = 35 degC (308 K)
Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)
refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe
ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and
Tm) were calculated using the nearest neighbor method3839
224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging
To determine the optimized conditions of stringency required to achieve selectivity for the
fully complementary oligonucleotide hybrids wash conditions were explored where selectivity
was controlled as a function of time and added formamide (vv) Paper substrates were washed
with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and
10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after
drying the paper substrates were imaged and the average intensity from reaction zones was
measured to calculate a quantitative ratiometric signal A wider range of wash conditions were
investigated for the sandwich assays because the energy associated with the PC hybrid MT probe
gQD gQD
gQD gQD
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
33
ndash WT Target was larger than other PC hybrids and could significantly shift conditions for
discrimination between FC and PC hybrids
Data from these wash condition experiments were summarized as heat map tables (see
Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich
formats wash conditions were explored with FC or PC targets For each probe sequence pairs of
heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets
were presented as green heat maps and PC targets were presented in red heat maps Wash
conditions suitable for assay development would have high signal from FC heat maps and very
low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white
ndash light red) for PC Wash conditions chosen for further investigations were then summarized in
Figure 14
2241 Labelled Target (Direct Format)
The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT
Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash
MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted
energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable
and to retain more signal under stringent wash conditions than PC hybrids
For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)
and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to
continue further investigations Similarly for MT probe the wash conditions offering the greatest
signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was
chosen as the wash condition to continue further investigations For WT probe the wash conditions
meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10
formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch
discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10
formamide at 5 and 10 min
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
34
Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids
WT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 108 plusmn 003 101 plusmn 003 094 plusmn 002
5 105 plusmn 003 096 plusmn 003 079 plusmn 002
75 102 plusmn 002 081 plusmn 003 080 plusmn 002
10 099 plusmn 001 07 plusmn 01 05 plusmn 01
Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids
WT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 098plusmn 002 020 plusmn 004 010 plusmn 001
5 092 plusmn 003 013 plusmn 002 000 plusmn 002
75 096 plusmn 002 012 plusmn 003 010 plusmn 002
10 093 plusmn 003 005 plusmn 001 002 plusmn 001
Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids
MT Probe - MT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 091 plusmn 005 104 plusmn 005 103 plusmn 002
5 087 plusmn 006 090 plusmn 001 068 plusmn 001
75 103 plusmn 003 091 plusmn 002 081 plusmn 003
10 101 plusmn 003 078 plusmn 003 062 plusmn 003
Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids
MT Probe - WT Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10
Amount of
Formamide
Added ( vv)
0 087 plusmn 002 022 plusmn 002 011 plusmn 001
5 086 plusmn 003 008 plusmn 003 005 plusmn 002
75 100 plusmn 003 007 plusmn 001 005 plusmn 002
10 095 plusmn 004 007 plusmn 001 004 plusmn 001
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
35
2242 Target Determination by Sandwich Assay
The process for determining the optimal wash conditions for sandwich assays was similar
to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in
for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids
Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target
hybrids FC hybrids were expected to be more stable and to retain more signal under stringent
wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target
hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more
stringent wash conditions to achieve discrimination of FC from PC sequences As with direct
assay discrimination of the FC hybrids from the PC hybrids required wash conditions where
ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise
of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids
(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal
wash condition to continue further investigations The wash conditions offering the greatest signal
for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen
as the optimal wash condition to continue further investigations
For MT probe the wash conditions meeting the criteria for mismatch discrimination are
more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic
treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide
at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min
meet the criteria for the assays Of the different wash conditions for MT probe only BB+5
formamide at 20 min met all the criteria because the BB+75 formamide and BB+10
formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT
probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures
of merit for the assays
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
36
Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids
WT Probe - WT
Targt
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004
125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006
25 074 plusmn 001 063 plusmn 003 061 plusmn 001 046 plusmn 003 040 plusmn 002
375 067 plusmn 003 056 plusmn 002 050 plusmn 003 043 plusmn 001 032 plusmn 003
5 062 plusmn 004 033 plusmn 006 028 plusmn 003 026 plusmn 002 020 plusmn 004
75 063 plusmn 002 019 plusmn 006 012 plusmn 004 013 plusmn 004 006 plusmn 002
10 052 plusmn 006 002 plusmn 004 002 plusmn 003 001 plusmn 003 000 plusmn 004
Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids
WT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 051 plusmn 006 002 plusmn 004 002 plusmn 006 004 plusmn 004 002 plusmn 003
125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004
25 062 plusmn 007 007 plusmn 005 003 plusmn 003 001 plusmn 004 002 plusmn 004
375 054 plusmn 004 003 plusmn 002 001 plusmn 003 000 plusmn 002 -001 plusmn 001
5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006
75 043 plusmn 007 -001 plusmn 003 000 plusmn 005 002 plusmn 002 -003 plusmn 002
10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004
Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids
MT Probe - MT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 065 plusmn 001 058 plusmn 007 058 plusmn 006 06 plusmn 01 055 plusmn 004
125 080 plusmn 008 076 plusmn 003 079 plusmn 005 071 plusmn 005 069 plusmn 004
25 07 plusmn 01 067 plusmn 006 071 plusmn 008 055 plusmn 006 056 plusmn 008
375 077 plusmn 007 071 plusmn 004 073 plusmn 003 070 plusmn 005 059 plusmn 006
5 072 plusmn 008 057 plusmn 006 049 plusmn 008 046 plusmn 007 037 plusmn 008
75 071 plusmn 002 044 plusmn 004 035 plusmn 003 026 plusmn 002 021 plusmn 004
10 073 plusmn 002 021 plusmn 005 01 plusmn 002 011 plusmn 004 010 plusmn 004
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
37
Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids
MT Probe - WT
Target
RG Ratio Signal
BB+X Wash Times (minutes)
0 5 10 15 20
Amount of
Formamide
Added (
vv)
0 06 plusmn 01 044 plusmn 008 04 plusmn 001 04 plusmn 01 036 plusmn 006
125 074 plusmn 005 068 plusmn 005 063 plusmn 004 046 plusmn 003 039 plusmn 001
25 07 plusmn 01 063 plusmn 008 058 plusmn 008 037 plusmn 005 028 plusmn 006
375 072 plusmn 002 062 plusmn 002 052 plusmn 002 033 plusmn 003 020 plusmn 003
5 07 plusmn 01 019 plusmn 002 016 plusmn 004 015 plusmn 002 006 plusmn 005
75 071 plusmn 001 011 plusmn 003 010 plusmn 004 006 plusmn 001 003 plusmn 003
10 070 plusmn 001 004 plusmn 002 002 plusmn 001 004 plusmn 001 003 plusmn 003
2243 Optimizing Wash Conditions for Selectivity
Of the various conditions investigated many provided for full discrimination of FC and
PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal
wash conditions for direct assays that provided the best resolution between FC and PC while
minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide
(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for
MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids
for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At
BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for
sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes
while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C
for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then
further investigated for the analytical figures of merit and performance in complex sample
matrices
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
38
Figure 14 Determination of optimal wash conditions for direct and sandwich assay
considered RG Ratios with variation of formamide concentration for wash times of 0 5 10
15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for
5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal
wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-
WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence
225 Analytical Figures of Merit
The performance of the bioassay was investigated in both direct and sandwich assay
formats and concentration-response curves are presented in Figure 15 Paper substrates were
washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times
of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich
assays Performance of the bioassays in the low pmol range is presented as insets for each of the
respective curves Regression analysis for the dataset was done to obtain the analytical figures of
merit which are presented in Table 11
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 15-02
00
02
04
06
08
Formamide in BB Wash (vv)
RG
Rati
oWT Target
MT Target
0 5 10 1500
05
10
15
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
0 5 10 1500
02
04
06
08
Formamide in BB Wash (vv)
RG
Ra
tio
WT Target
MT Target
gQD
gQD
gQD
gQD
gQD
gQD
gQD
gQD
Optimized Condition (Direct Assay) BB+10F for 5 mins
Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)
C D
A B
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
39
Figure 15 Concentration-response curves showing the RG ratiometric response of the
direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for
determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used
for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for
determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT
probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled
reporters The RG ratiometric response of the direct assay at the low pmol concentration
range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe
conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT
probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar
represents one standard deviation for n=4 replicates
The response of the WT and MT direct assays was similar with sensitivity (slope of
response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two
orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol
for WT and MT probes respectively This consistency in analytical performance reflects the
similar ∆G and Tm for the two FC and PC hybrids
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
40
Table 11 Analytical Performance Direct and Sandwich Bioassays
Assay
Format
Probe Slope of
Calibration
Curve
r2 LOD LOQ Linear
Range
(pmol)
Direct
Assay
WT 03145 09857 215 fmol 650 fmol 03 ndash 15
MT 03147 09680 285 fmol 865 fmol 03 ndash 15
Sandwich
Assay
WT 00486 09934 422 fmol 128 pmol 04 ndash 20
MT 00285 09779 145 pmol 438 pmol 15 ndash 20
The sandwich assay response of WT and MT was found to vary with WT probes having
double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a
larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical
performance are also consistent with the thermodynamic stabilities of the various hybrids MT
probes were required to undergo washes of higher stringency and thus a larger proportion of the
FC was lost Quantification of the analytical parameters was accomplished using only WT or MT
targets However the discrimination of targets in mixtures is also of importance
226 Selectivity for Mixtures of WT and MT Targets
Clinical samples of oligonucleotides are expected to be composed of gene sequences of
WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT
sequences must therefore be evaluated Selectivity assays were determined in direct assay format
and signal from digital images was measured pre- and post- formamide washing Samples of 24
pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets
(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done
using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with
0 ndash 48 pmol samples of CFTR PC targets
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
41
Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes
and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined
using background corrected RG ratio plots for hybridization of gQD-probe conjugates with
Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled
targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the
hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-
wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in
Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error
bars represent one standard deviation for n = 4 replicates
It was found that for both direct and sandwich assays in pre-wash WT and MT signals
showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct
assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from
0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids
Post-wash it was found that there was no contribution of signal from the addition of PC targets to
either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
42
assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable
stringency control can obviate false positives in mixtures of WT and MT probes
227 Paper-based Assay Response for Complex Sample Matrices
The performances of the assays were investigated for samples that contained bovine serum
albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp
fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR
WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol
concentration for sandwich assay The resulting RG ratios from direct hybridization assays
(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates
respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)
for WT and MT probes respectively
Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in
complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or
BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates
and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to
direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
43
collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars
represent one standard deviation for n = 4 replicates
High selectivity was retained for all hybridization assays in both direct and sandwich
format with the signal from NC and PC hybrids being within the experimental error Thus the
interfering effects of these sample matrices did not compromise the performance of either direct
or sandwich assays
228 Blind Assay for Detection and Quantification of CFTR Target Mixes
The performances of the direct and sandwich assays were investigated with a blind assay
experiment to confirm that the specific wash conditions in this thesis could be used for
determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence
Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were
used in particular because these are the expected combinations of oligonucleotides from clinical
samples The blind assays were prepared with external assistance such that sample identities and
concentration were unknown to the assayer Samples were prepared in BBS buffer with a final
concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions
were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for
sample identification Signal from the assays and subsequent identification of samples were found
to be in agreement and within experimental error supporting applicability of this technology for
clinical application (see Table 12) All spiked samples were correctly identified by the assayer
and signals generated from assays were within the dynamic range of the assay
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
44
Table 12 Blind Assay for Direct and Sandwich Assays
Assay Format Blind
Sample
Spiked
Samples
Signal Sample
Identification WT assay MT assay
Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT
2 WT and MT 049 plusmn 001 058 plusmn 004 Mix
3 MT only 000 plusmn 002 065 plusmn 006 MT
4 MT only 001 plusmn 003 043 plusmn 002 MT
Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT
2 WT and MT 024 plusmn 003 043 plusmn 003 Mix
3 WT and MT 025 plusmn 002 040 plusmn 001 Mix
4 MT only 003 plusmn 002 035 plusmn 005 MT
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
45
Chapter 3
Conclusion and Future Work
Fluorescence determination in a paper substrate of a predominant genetic marker for cystic
fibrosis has been explored This involves distinction between a mutant form and wild type
oligonucleotide sequence either of which could be present individually or in mixture in clinical
samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3
molecular fluorophore as an acceptor has provided for two assays methods One method relied on
labelled oligonucleotide target as commonly produced during enzyme amplification Another
method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets
Analytical performance was primarily based on selective melting of undesired hybrids and
sufficient stringency control was possible to provide reliable detection of targets even in samples
that contained substantial quantities of protein and nucleic acid as interferents Despite the
performance differences due to thermodynamic stabilities of hybrids formed from two
oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that
both direct and sandwich assays could be implemented to distinguish between wild type and
mutant type samples
Of the two hybridization formats direct assay was observed to have better analytical
figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which
had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on
the order of five minutes with direct assay using more stringent wash conditions than sandwich
assay However the MT variant for sandwich assay was found to have a higher LOD and smaller
dynamic range than other sequences Wash times for the MT sandwich assay was four times as
long as WT and direct assays limiting the throughput of this assay in sandwich format Taking
these facts into account sandwich assay is still better suited for further development of this
technology than direct assay Sandwich assays can be incorporated with ease to different types of
amplification techniques when compared with direct assay which requires labelled nucleotides
limiting the options available for amplification
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
46
31 Future Directions
There are many requirements that need to be addressed for the application of this screening
technologies for the point-of-care The work in this thesis focused primarily on the detection of
targets related to Cystic Fibrosis but the sample processing target extraction target amplification
and clinical validation still need to be addressed Samples for POC genetic testing will need to be
processed without the use of large laboratory instruments because the technology for a device must
be portable and low cost Extraction and amplification of targets will also be required due to the
low number of targets present in samples
The two most likely applications for this technology are the incorporation of paper-based
test strips for new born screening of infants7-10 and general screening for CF genes of adult
patients The implementation of multi-level NBS programs is relatively new and is based firstly
on a heel prick blood test followed by a larger volume blood and sweat test The small volume of
blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique
to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker
for analysis with the paper-based test strip49 Amplification techniques like PCR and
tHDA7482 have been shown to detect these levels of genetic material and would be required for
further application of the proposed paper-based technology Blood tests for adults could include
screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of
blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic
material49 These larger amounts of nucleic acids can be amplified using simpler technology
associated with isothermal enzymatic methods given that exponential amplification may not be
essential to achieve sufficient signal from hybridization assays
Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming
low target numbers because it eliminates the need for temperature control modules currently
required for enzyme-based amplification The lack of specialized equipment makes isothermal
techniques field portable and POC available Two popular isothermal techniques that are being
translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and
recombinase polymerase amplification (RPA)51 These technologies will also require clinical
validation with real patient samples at the POC for further application
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
47
References
(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical
Application Nat Rev Genet 2015 16 (1) 45ndash56
(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking
and Jumping Science 1989 245 (4922) 1059ndash1065
(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash
1904
(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012
(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science
1989 245 (4922) 1073ndash1080
(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and
Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073
(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic
Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661
(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015
136 (6) 1062ndash1072
(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities
J Pediatr 2008 153 (3) 308ndash313
(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic
Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J
Hum Genet 2009 17 (1) 51ndash65
(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests
httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics
ucm330711htm (accessed Feb 22 2018)
(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and
Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008
80 (10) 3699ndash3707
(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic
Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)
19606ndash19611
(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-
Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45
(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and
Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York
2002
(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563
(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and
Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New
York 2002
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
48
(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W
H Freeman and Company 2014 pp 493ndash525
(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the
Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82
(1) 3ndash10
(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash
288
(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol
Chem 1919 40 415ndash424
(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature
1953 171 (4356) 740ndash741
(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids
Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740
(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for
Deoxyribose Nucleic Acid Nature 1953 171 737ndash738
(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual
Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169
(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic
Degradation Experientia 1950 6 201ndash209
(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad
Sci 1958 44 (7) 671ndash682
(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein
In Molecular Biology of the Cell 5th Edition Garland Science New York 2002
(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of
the Human Genome Nature 2001 409 (6822) 860ndash921
(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)
100
(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable
DNA Polymerase Science 1988 239 (4839) 487ndash491
(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-
Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F
Academic Press 1987 Vol 155 pp 335ndash350
(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res
2000 28 (12) e63
(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on
Biochips Anal Biochem 2010 397 (1) 132ndash134
(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev
Biophys Biomol Struct 2004 33 415ndash440
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
49
(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability
from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750
(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor
Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)
3555ndash3562
(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor
Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT
Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477
(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC
Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998
37 (26) 9435ndash9444
(42) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in
DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594
(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal
Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping
Assays Nucleic Acids Res 2008 36 (2) e10
(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and
Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl
Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11
(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability
Nucleic Acids Res 1996 24 (11) 2095ndash2103
(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low
Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114
(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents
Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys
1993 99 (1) 113ndash125
(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to
Methods and Applications Academic Press 2012
(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection
Electroanalysis 1996 8 (1) 15ndash19
(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification
EMBO Rep 2004 5 (8) 795ndash800
(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using
Recombination Proteins PLoS Biol 2006 4 (7) e204
(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat
Biotechnol 2003 21 (10) 1192ndash1199
(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA
Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
50
(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical
Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26
(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and
Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem
B 1997 101 (46) 9463ndash9475
(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent
Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol
2002 13 (1) 40ndash46
(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters
(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496
(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly
Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J
Am Chem Soc 1993 115 (19) 8706ndash8715
(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996
271 (5251) 933ndash937
(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots
in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837
(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated
CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871
(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection Science 1998 281 (5385) 2016ndash2018
(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA
Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123
(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as
Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett
2001 1 (9) 469ndash474
(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive
Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86
(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic
Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336
(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886
(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761
(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry
Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060
(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of
Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing
Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25
(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York
2006
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
51
(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash
1395
(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-
VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013
(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent
Nanoparticles Analyst 2016 141 (10) 2838ndash2860
(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates
A Versatile Platform for Biosensing Energy Harvesting and Other Developing
Applications Chem Rev 2017 117 (2) 536ndash711
(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections
(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6
(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple
Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash
7095
(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper
Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998
(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash
4774
(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications
Anal Chem 2016
(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on
Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837
(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric
Fluorescence Transduction by Hybridization after Isothermal Amplification for
Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based
Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165
(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular
Diagnostics Anal Chem 2015 87 (15) 7595ndash7601
(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection
of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121
(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based
Microfluidics Anal Chem 2009 81 (14) 5821ndash5826
(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay
Anal Chem 2013 85 (24) 11691ndash11694
(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid
Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence
Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867
(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection
Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
52
(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific
and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based
Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947
(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive
and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic
Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem
Int Ed 2012 51 (20) 4896ndash4900
(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple
Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713
(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care
Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251
(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces
for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84
(7) 3311ndash3317
(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid
Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As
Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash
7511
(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped
LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917
(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer
Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and
Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8
(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic
Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655
(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic
Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform
Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339
(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers
for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)
2951ndash2958
(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-
Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization
Lab Chip 2013 13 (19) 3945ndash3955
(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash
1582
(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand
Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash
1305
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures
53
Copyright Acknowledgements
Copyright permissions have been reported in the caption for relevant figures