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Antibody microarrays visualized bycarbon nanoparticles - from R to D
Aart van Amerongen
Scienion Workshop, Berlin, 21/22-10-2010
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Sciences group from Wageningen University and
Research Centre (WUR) Combination of university departments and a not-for-profit
contract research organisation (CRO)
CRO: Wageningen UR Food & Biobased Research
Research on agro-biopolymers, plants, crops and food fromprimary production up to end-products
> 70% of overall budget is result of acquisition of projects
'Customers': SMEs, multinationals, governments, EU, ......
Agrotechnology & Food Sciences Group
Business units:
Food, Freshness and Chains
Biobased Products
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Content
General introduction in protein adsorption
Influence of parameters on functionality of printed proteins
Buffer composition (and humidity)
Substrate hydrophobicity (glass slides)
Increase of surface area to volume ratio
Nitrocellulose on slides
Colloidal carbon nanoparticles
Examples of using carbon nanoparticles; from R to D
VTEC diagnostics Malaria diagnostics
New assay formats with carbon nanoparticles
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Multi-analyte diagnostics
Despite its high potential protein multi-analyte assays are
still not emerging tools in the regular diagnostic field Limited presence due to factors such as the lack of sufficient
biological recognition elements (e.g., antibodies) and/or theirsensitivity, specificity, and cross-reactivity, the lack of
integrated systems that include fluid handling, samplepreparation and signal processing and inacceptablebackground signals
To apply protein multi-analyte assays many problems
have still to be overcome to end up with validated in vitrodiagnostics as indicated by Hartmann et al. (2009)
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Multi-analyte diagnostics
Binding of oligonucleotides to solid phase materials
DNA microarrays:
Only 4 building blocks
Individual probes have the same orientation
For each oligonucleotide probe the chemical interaction with
the surface is identical
covalent chemical bond
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Multi-analyte diagnostics
Binding of proteins to solid phase materials
Protein microarrays:
Proteins consist of over 20 different building blocks (aminoacids) with very different characteristics
Each protein has a unique structure, pI, (micro-)surface
characteristic, etc. Consequently, in the ideal situation each protein would need
dedicated conditions for optimal binding onto solid phasematerials
From a functional point of view the adsorption of proteinsonto surfaces, membranes, or nanoparticles is even morecrucial
Arnoldus W.P. Vermeer, Willem Norde, Aart van Amerongen
(2000) The unfolding / denaturation of IgG of isotype 2b and itsFAB and FC fragments. Biophysical Journal 79, 2150-2154
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Multi-analyte diagnostics
Binding of proteins to solid phase materials
Critical factors that influence adsorption:
Application buffer
ionic strength pH
composition co-precipitating agents
Type of membrane / surface
Application system
Surface potential plot onsolvent accessable surface;blue is positive and red is
negative charge
Surface potential plot onsolvent accessable surface;blue is positive and red is
negative charge
Arnoldus W.P. Vermeer, Willem Norde, Aart van Amerongen
(2000) The unfolding / denaturation of IgG of isotype 2b and itsFAB and FC fragments. Biophysical Journal 79, 2150-2154
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Multi-analyte diagnostics
Binding of proteins to solid phase materials
Result if using one particular set of binding conditions:
In general, functionality (binding or enzymatic activity) of theindividual proteins will not be optimal
IgG1 model; electric potential plotIgG1 model; electric potential plotphysical or chemical bond
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Multi-analyte diagnostics
Binding of proteins to solid phase materials
Two-Compartment Model (TCM)
Originally proposed for the analysis of mass-transport limitedbiomolecular interactions in the Biacore instruments
Compartments:
1. Transport of the analyte from the bulkcompartment to the surface reaction area
2. Subsequent binding process in reactioncompartment
Analytes
Binding molecules Compartment 2
Compartment 1Analytes
Binding molecules Compartment 2
Compartment 1
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Multi-analyte diagnostics
Binding of proteins to solid phase materials
TCM was modified for the analysis of interactions in themicroarray format by Kusnezow et al.*
Conclusion:
Strong limitation by mass transport to the surface
Kinetics may be slowed down as much as hundreds oftimes compared to the solution kinetics
Very important to address this point (e.g., by shaking and/orincreased incubation temperature)
*: see, e.g., J.Chem.Phys. (2005); Proteomics (2006);Mol.Cell.Proteomics (2006); Expert Rev.Mol.Diagn (2006)
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Multi-analyte diagnostics
Parameters that influence multi-analyte assay results
Stirring and Geometry of the incubation chamber
Minimally permissible sample volume
Binding site density / Antibody spotting concentrations
Surface chemistries
Viscosity of sample and buffers
Spotting pattern
Concentration of analyte in the sample
Incubation times
Washing stages and Signal generation
Non-specific binding / Background
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Influence of various parameters onthe functionality of printed proteins
Non-contact inkjet printed proteinsSTW project 10095: Biomolecules Substrate Topographyof Inkjet Printed Structures (Bio-STIPS)
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Bio-STIPS
Background:
In many evaporation processes of practical importance such asthe evaporation of solvents filled with polymer or in DNA andprotein microarrays, the viscosity of the fluid increasessubstantially with solute concentration, and hence during theevaporation process
This has a large influence on the shape of the deposition andthe distribution of (bio)molecules on the substrate, while thefunctioning of e.g. the microarrays is critically dependent on thisdistribution
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Bio-STIPS participants
Scientific collaborators:
Eindhoven University of Technology, Dept. of MechanicalEngineering (coordinator dr. Hans Kuerten)
Wageningen University, Laboratory of Physical Chemistry andColloid Science (Prof.dr. Willem Norde)
Wageningen UR Food & Biobased Research, BiomolecularSensing & Diagnostics (dr. Aart van Amerongen)
User committee:
Philips Research, Oc Technologies NV, Chematronics B.V.,
Animal Health Service Deventer, PamGene International B.V.,Scienion AG, Eindhoven University of Technology (MesoscopicTransport Phenomena), Friedrich-Schiller-Universitt Jena(Lab. Organic & Macromolecular Chemistry)
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Bio-STIPS
Theoretical science (Eindhoven University of Technology)
Development of an extended physical model for the calculationof the thickness of deposition that results after evaporation of asolvent from a droplet and of the distribution of (bio)moleculeson/in the substrate
'Extended' means that all physical phenomenathat play animportant role in practical applicationswill be taken into account
Subsequently, this physical model will be implemented in anumerical computer program
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Bio-STIPS
Theoretical science (Eindhoven University of Technology)
The complexity of the physical phenomena involved, inparticular the interaction between the solute molecules and thesubstrate, makes experimental validation a necessity
Moreover, relevant physical parameters, such as(concentration-dependent) viscosity, diffusivity, evaporationvelocity and porosity of the substrate, are needed for thedevelopment of the model
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Bio-STIPS
Experimental science (Wageningen University)
Experimental techniques on evaporating droplets
The influence of the substrate surface and buffer compositionon the orientation and conformation and, consequently, thefunctionality of biomolecules is of crucial importance
Model proteins will be deposited and the orientation and/orconformation of these proteins will be studied by applyingsurface epitope-/region-specific antibodies
Experimental techniques include atomic force microscopy,interference contrast microscopy, (confocal) fluorescent
microscopy, reflectometry, contact angle experiments,fluorescence spectroscopy and methods based on affinitybinding in the layer
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Scienion S3 microarrayer
Non-contact arrayer: up to 8 nozzles
Piezo-driven dispenser
Droplets of down to 200 pL can be printed
Diameter of spots is 200 m
Humidity-controlled
3 mm
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Influence of various parameters onthe functionality of printed proteins
Buffer composition (and Humidity)
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Influence of immobilization conditions
Model: BSA-biotin
Substrate - Greiner HTA polystyrene substrate slides
ELISA plate-like material !
Buffers - PBS (pH 7.4), Carbonate Buffer (CB, pH 9.6)
Drying - RT at 22% and 70% humidity
Results following specific staining with carbon nanoparticleswith neutravidin after printing in: PBS ~70% CB ~70%
Better intensities after printingBSA-biotin in carbonate buffer
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Reflectometry experiments with BSA-biotin
Reflectometry is an optical technique for the determination
of the adsorption of molecules from solution onto amacroscopically flat substrate
0S
SQf mg/m
2
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Reflectometry experiments with BSA-biotin
Results on HTA polystyrene surfaces:
BSA-bt in PBS BSA-bt in CB
Higher affinity of BSA-biotin for polystyrene surface ifdissolved in PBS buffer
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Atomic Force Microscopy of BSA-biotin spots
Experimental set up:
Atomic Force Microscopy of microarray spots
Incubation with buffer
Resembling blocking/washing step Incubation with fluorescent streptavidin
Atomic Force Microscopy:
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Atomic Force Microscopy of BSA-biotin spots
BSA in PBS (250 g/mL; 1 droplet)
Expected: smooth spot, uniformly distributed BSA-biotinmolecules
Printed droplet Fluid evaporation Biomolecules onsurface
Biomolecules onsurface following first
wash / incubation
AFM pictures
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Atomic Force Microscopy of BSA-biotin spots
BSA in PBS (250 g/mL; 1 droplet)
Observed:
Height of structures: up to 1000 nm
Monolayer of BSA would be 10 nm
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Specific staining with fluorescent streptavidin
Confocal Laser Scanning Microscopy
No signal in areas where structures were removed duringblocking/washing step => even no monolayer of BSA !?
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Influence of immobilization conditions
Model BSA-biotin (250g/mL)
Substrate: HTA polystyrene slide Spotting buffer : Carbonate buffer (pH 9.6)
Average height of ~150nm
3D view Height data
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Influence of immobilization conditions
Much better spot coverage in carbonate buffer than in PBS
IgG showed similar results on HTA polystyrene slides:
AFM- 3D view AFM-Height data
PBS(pH 7.4)
CB(pH 9.6)
~ 400 nm
~ 240 nm
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PBS
(pH 7.4)
PB
(pH 7.4)
CB
(pH 9.6)
BSA-bt
200g/mL
IgG-bt
200g/mL
Influence of immobilization conditions
Omitting NaCl from PBS resulted in better / good spots
Imaging method: Tapping mode Substrate: HTA polystyrene slide
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Example of influence of drying conditions
Model: BSA-biotin
Substrate - HTA polystyrene substrate
Buffer - carbonate buffer (pH 9.6)
Drying - RT at 22% and 70% humidity
0
2e+5
4e+5
6e+5
2
4
68
10
12
14
161820
24
68
1012
1416
18
Z
Data
XD
ata
YData
[BSA-bt]=250g/mL (2 drops)
0
2e+5
4e+5
6e+5
0
2e+5
4e+5
6e+5
2
4
68
10
12
14
161820
24
68
1012
141618
Z
Data
XD
ata
YData
[BSA-bt]=250g/mL (2 drops)
0
2e+5
4e+5
6e+5
~22% ~70%
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Influence of various parameters onthe functionality of printed proteins
Substrate hydrophobicity
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Protein binding to substrates
For the protein in aqueous solution, the hydrophilic side
chains are presented at the outside and the hydrophobic tothe inner of the protein to avoid contact with water
Attached to a surface, new interactions become possiblefor the protein; in the first step, the protein rearranges its
structure to reach an energetically advantageousconformation
Because of interactions with adjacent protein moleculesand the surface, additional rearrangements can occur,
resulting in a partly or totally denaturated protein Are these adsorbed protein molecules still functional ?
Christine Mller, Anne Lders, Wiebke Hoth-Hannig, Matthias Hannig,and Christiane Ziegler. Initial bioadhesion on dental materials as a
function of contact time, pH, surface wettability, and isoelectric point.Langmuir 26 (2010) 41364141
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Protein binding to substrates
Adsorption model:
From: Mondon M.Untersuchungen zur Proteinadsorptionaufmedizinisch relevanten Oberflchen mit Rasterkraft-
spektroskopie und dynamischer Kontaktwinkelanalyse.Ph.D. Thesis, University of Kaiserslautern, 2002
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Substrate hydrophobicity
Substrate hydrophobicity influences
the deposition of protein molecules
On hydrophilic surfaces (A)droplets spread
Spreading of a droplet inducesinternal advection andenrichment of dissolved proteinmolecules at the dropletperimeter ('donut-structure')
On hydrophobic surfaces (B) droplets contract The advective situation in a high contact angle drying droplet
results in a homogeneous distribution of analyte
Anton Ressini, Gyrgy Marko-Varga and Thomas Laurell. Poroussilicon protein microarray technology and ultra-/superhydrophobic
states for improved bioanalytical readout. Biotechnology AnnualReview 13 (2007)
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Contact angle of water droplet on silanized glass
Material Silane derivativeContactangle
2 L droplet in Goniometer
Glass < 10
CPTES3-Cyano Propyl TriethoxySilane
~ 49
GPTMS 3-Glycidyloxy PropylTrimethoxy Silane
~ 61
PhECS Phenyl Ethyl Chloro Silane ~ 75
HMDS Hexa Methyl DiSilazane ~ 88
DCDMS DiChloro Dimethyl Silane ~ 102
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Substrate hydrophobicity
Influence on protein distribution
Printed: IgG molecule with biotin attached
Specific staining by streptavidin with attachedfluorochrome Alexa-633
Confocal Laser Scanning Microscopy
Untreated GPTMS DCDMS
< 10 ~ 61 ~ 102
CLSM
Alexa-633-Streptavidin
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Increase of surface area tovolume ratio
Nitrocellulose substrate and Carbon nanoparticles
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Increase of surface area to volume ratio
Solutions chosen:
Use of nitrocellulose membranes Lateral and cross-flow immunoassays
Microarray (lateral flow) immunoassays
Microarray approach:
increasing sensitivity bydecreasing spot diameter
Examples with carbonnanoparticles
Verotoxigenic EscherichiacoliO157
Malaria species detection
Nexterion
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Colloidal carbon nanoparticles
Introduction to carbon nanoparticles
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Carbon nanoparticles
Characteristics:
Clusters of primary particles => 50 - 400 nm, depending on thecarbon pigment used, with or without surfactants / detergents
Advantage in terms of sensitivity and Hook-effect
----- 10 m
---- 1 m
----- 100 nm
Sensitivity of lateral flow labels: Julian Gordon and Gerd Michel; Foundation
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Sensitivity of lateral flow labels:
Carbon nanoparticles in top-10; low picomolar detection
Analytical sensitivity limits for lateral flow immunoassays. Clinical
Chemistry 54 (2008) 1250-1251. Including Table 1 + SupplementFindDiagnosticsOrg (www.clinchem.org/cgi/data/clinchem.2007.102491/DC1/1)
for Innovative New Diagnostics (FIND)
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Lateral Flow and Microarray ImmunoAssays
Detection targets
LFIA / MIA: proteins, microbial cells, chemical components,carbohydrates, ......
Nucleic Acids: NALFIA / NAMIA: specific DNA / RNA amplicons(tags incorporated in product, or via tag-labelled probes)
Results:
LFIA / MIA =>
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Nucleic Acid LFIA (NALFIA) or MIA (NAMIA)
Rapid detection of genetic material
Examples: micro-organisms (human, veterinary, feed / foodpathogens)
Procedure (PCR procedure down to 15 minutes):
double-strand
double-tagged
primer 1
primer 2
tag 1
tag 2
amplification withtemplate DNA
(e.g. PCR protocol) amplicon
Alternative procedure: amplification to single chainamplicons that are hybridized to tag-labelled probes
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Tags used in NALFIA / NAMIA
Examples of tags used:
Forward primer tags: DNP (dinitrophenol), TXR (texas red),FAM (fluorescein carboxy-amido), Cy5 (cyanine), DIG(digoxigenin), TAM (TAMRA; carboxytetramethylrhodamine)
Detection system is generic:
Carbon nanoparticles always coated withneutravidin
Antibodies spotted onto the membranerecognize one of the tags
Specificity is in the combination offorward primer and tag
Any target can be assigned to a particularline (NALFIA) or spot (NAMIA)
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Examples of using carbon nanoparticles
VTEC diagnostics:
Rapid, multi-analyte assays for the detection of genes codingfor verotoxigenic Escherichia coliO157 (VTEC) and four
virulence factorsCollaboration in the context of the Diagnostics Platform of Wageningen UR(A. de Boer, K. Maassen, F.J. van der Wal)
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Verotoxigenic E. coli (VTEC)
Also called Enterohemorrhagic E. coli
Symptoms Gastroenteritis
Haemorrhagic colitis (HC)
Haemolytic uraemic syndrome (HUS)
Reservoirs Cattle, sheep
Transmission by consumption of
Undercooked meat
Unpasteurized dairy products
Contaminated water or vegetables
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Verotoxigenic E. coli (VTEC)
Virulence factors:
Elaborate phage-encoded cytotoxins: verotoxins vt1 vt2
Intimin protein (VTEC attachement to intestine epithelial cells)
eae Enterohaemolysin
ehec
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VTEC research goals
Development of two rapid molecular biological methods
with carbon nanoparticles as signal labels to detect thepresence of four 'classical' virulence factors (vt1, vt2, eae,ehec) and a 16S control specific for E. coli
Methods:
Nucleic Acid Lateral Flow ImmunoAssay (NALFIA)
Nucleic Acid Microarray ImmunoAssay (NAMIA)
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PCR for VTEC assays
Name Type Explaining Pimer sequence Fragment Label
vtx1 vt1-F Verotoxin 1 5'-GGATAATTTGTTTGCAGTTGATGTC-3' 107 bp Texas Red
vt1-R 5'-CAAATCCTGTCACATATAAATTATTTCGT-3' biotinvtx2b vt2-F Verotoxin 2 5'-GGGCAGTTATTTTGCTGTGGA-3' 130 bp FITC
Vt2-R 5'-GAAAGTATTTGTTGCCGTATTAACGA-3' biotin
eae eae-F Intiminadhesion
5'-CATTGATCAGGATTTTTCTGGTGATA-3 102 bp DIGeae-R 5'-CTCATGCGGAAATAGCCGTTA-3 biotin
ehxA ehec-F Hemolysin 5'-CGTTAAGGAACAGGAGGTGTCAGTA-3' 142 bp DMP
ehec-R 5'-ATCATGTTTTCCGCCAATGAG-3' biotin
16S Hui -F Small ribosomesubunit
5'-CATGCCGCGTGTATGAAGAA-3' 96 bp Cy5
hui-R 5'-CGGGTAACGTCAATGAGCAAA-3' biotin
PCR reaction (for all primers)PCR program Reagents
Temp time Water 5 L
98 C 30 s Phire Buffer 5x 4 L final [MgCl2] = 1,5 mM
98 C 5 s dNTP 5 L final [dNTP] = 0,25 M
61 C 5 s 30 cycles Primers (10 M) 1,8 L final [primer] = 0,9 M (each)72 C 5 s Phire polymerase 0,4 L
72 C 1 m Template 2 L
4 C Total volume 20 L
30 min
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Results of VTEC NALFIA and NAMIA
Nucleic acid-based detection of verotoxigenic E.coliO157
and 4 virulence factors; trial with 48 field-derived E.colisamples
NALFIA:
B vt1 vt2 ehec eae 16S all
control
-TXR (vt1)
-FITC (vt2)
-DNP (ehec)-DIG (eae)
-Cy5 (16S)
Line/Gene Antibody mg/mL
control IgG-biotin 200
vt1 Texas Res 200vt2 FITC 50
ehec DNP 200
eae DIG 20016S Cy5 800
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Results of VTEC NALFIA and NAMIA
Nucleic acid-based detection of verotoxigenic E.coliO157
and 4 virulence factors; trial with 48 field-derived E.colisamples
NAMIA:
Average pixel grey levels were automatically calculated andscored positive if > 3 x background SD
ehec
blank
control
eae
16S
vt2
vt1
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VTEC NALFIA and NAMIA compared to Q-PCR
Vt1 Vt2 eae ehec 16S
Nalfia Namia Nalfia Namia Nalfia Namia Nalfia Namia Nalfia Namia
Sensitivity (%) 85.0 85.0 100.0 100.0 100.0 82.6 96.9 96.9 100.0 100.0
Specificity (%) 96.4 100.0 88.9 100.0 100.0 100.0 100.0 93.7 100.0 100.0
Efficiency (%) 91.7 93.8 95.8 100.0 100.0 91.7 97.9 95.8 100.0 100.0
Comparison:
NALFIA manuscript accepted by Analytical and
Bioanalytical Chemistry, in press Microarray manuscript in preparation
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Examples of using carbon nanoparticles
Malaria diagnostics:
Species identification and SNPs analysis related toartemisinin drug combination therapy resistance
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Malaria diagnostics
Multi-drug resistance in malaria under combination
therapy: Assessment of specific markers and developmentof innovative, rapid and simple diagnostics
Project acronym: MALACTRES
EU7 - HEALTH project
2008 - 2012BeneficiaryNumber
Beneficiary name Beneficiaryshort name
Country1(coordinator)
Royal Tropical Institute KIT The Netherlands
2 Institute of Tropical Medicine ITM Belgium
3 London School of Hygiene and TropicalMedicine
LSHTM United Kingdom
4 Biomolecular Sensing & Diagnostics,
Wageningen University and Research Centre
AFI The Netherlands
5 Forsite Diagnostics Limited FDL United Kingdom
6 Tropical Diseases Research Group, Faculty ofPharmacy, University of Benin
UNIBEN Nigeria
7 Centre Muraz CM Burkina Faso
8 Kilimanjaro Christian Medical Centre KCMC Tanzania
Participants:
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Malaria diagnostics
Facilities in Africa (Burkina Faso)
Research institute in Bobo Dioulasso
Hospital near Ouagadougou
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Malaria diagnostics - NALFIA
Development of point-of-care diagnostics
Nucleic Acid Lateral Flow ImmunoAssay (NALFIA) Nucleic Acid Microarray ImmunoAssay (NAMIA)
Two-line NALFIA
Test control line
Pan-Plasmodium line
Recognizing all Plasmodiumspecies
Trial in Kenya
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Malaria diagnostics - NALFIA
NALFIA compared with microscopy (gold standard) and
agarose gel electrophoresis
Petra F. Mens, Aart van Amerongen, Patrick Sawa, Piet A. Kager, HenkD.F.H. Schallig. Molecular diagnosis of malaria in the field: development of anovel 1-step nucleic acid lateral flow immunoassay for the detection of all 4
human Plasmodium spp. and its evaluation in Mbita, Kenya. DiagnosticMicrobiology and Infectious Disease 61 (2008) 421-427
650 clinically suspected malariacases
NALFIA detection limit: 0.3 - 3parasites per L
More sensitive than microscopy
10-fold more sensitive than gelelectrophoresis
Excellent agreement with gelelectrophoresis
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Malaria diagnostics - NALFIA
Recent trial in Burkina Faso
Four-lines NALFIA (GAPDH,Plasmodium falciparum, Plasmodiumvivax, Pan-Plasmodium)
Examples of the 4-lines PCR-NALFIA with a P.falciparum (left)
and a P.vivax specific sample
Initial statistics:
Plasmodium falciparumand Pan-Plasmodium excellent sensitivity
(97 and 98%, respectively)
Plasmodium vivaxsensitivity only57% (but: Pan-Plasmodium OK)
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Malaria diagnostics - Microarray method
Anti-tag antibodies printed on Nexterion slides:
128, 320 and 780 ng/l anti-TexasRed 32, 80 and 200 ng/l anti-Dig 16, 40 and 100 ng/l anti-FITC 64, 160 and 380 ng/l anti-DNP
Microtiter plate-sized holder for 4slides; 64 samples
M l i di i Mi h d
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Malaria diagnostics - Microarray method
Added reagents (one-step incubation; 1 h)
PCR (multiplex): 4 L Carbon conjugate: 5 L
Neutravidin - Alkaline phosphatase (AP) fusion protein
Following incubation the lower halfof the slide was incubated with APsubstrate (10 min incubation)
Precipitating dye increases signal
M l i di i Mi h d
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Malaria diagnostics - Microarray method
NAMIA: Microarray detection of malaria-specific amplicons
Staining by carbon nanoparticles (upper 8 pads; 1 hour) Subsequent and additional staining by alkaline phosphatase
substrate conversion (lower 8 pads; + 10 min)
Mi Vi i l i f
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MicroVigene microarray analysis software
Following digitization of the slides (16 microarrays) by
flatbed scanning or digital camera the softwareautomatically positions the grid on the scanned tif-file
Subsequently, data processing is done in less than 5 min After copying the raw data into an Excel template sheet
the results from all 16 pads are available immediately
M l i di ti Mi th d
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Malaria diagnostics - Microarray method
Single-laboratory trial
Microarray design Spots available for other
targets like SNPs
Procedure: Carbon nanoparticles (1 hour) followed by 3 shortwash steps with running buffer, alkaline phosphatase substrate(10 min) and a short wash step with MQ
M l i di ti Mi th d
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GAPDH
Concentration of individual amplicons: 0.8 pmol
Pan
vivax
falc
GAPDH
falc
vivax
Pan
GAPDH
vivax
Pan
GAPDH
falc
Pan
PCR withouttemplate
Concentrationof amplicons incombination:
0.2 pmol
Concentration ofamplicons in
combinations: 0.3 pmol
Of all fourPCR
reactions:1 L
Amplicons:
Malaria diagnostics - Microarray method
Nexterion slide; various combinations of amplicons
Microarray trial in Africa scheduled in spring 2011
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New assay formats with carbon
nanoparticles
Microarrays in wells of ELISA plates Lateral flow microarray immunoassay (LMIA)
N f t ith b l b lli
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New assay formats with carbon labelling
Microarrays in wells of microtiter plates
As compared to conventional ELISA: multi-analyte assay with the same samplevolume
Well-developed assay platform (> 50 years)
Skills to execute assay are broadly present Equipment for automated handling
available (robotic liquid handling, washing,staining, data recording and processing)
Needed:
On-deck holder for microtiter plates Reader based on the scanning principle
(colorimetric, fluorometric)
N f t ith b l b lli
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New assay formats with carbon labelling
Lateral flow Microarray ImmunoAssay (LMIA)
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Concluding remarks
Carbon nanoparticles are excellent labels for rapid lateral
flow and microarray methods
The signal-to-noise ratio of these nanoparticles enableslow picomolar detection by visual inspection
Digitization and quantification of the results can beautomated
Rapid Methods Europe 7: 24-26 January 2011, Noordwijkerhout, The Netherlands
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Thank you for your attention !
[email protected] Wageningen UR
Acknowledgements: Ren Achterberg, Kitty Maassen and Fimme Jan van der Wal (Wageningen UR CVI)
Marjo Koets, Antoine Moers, Liyakat Mujawar, Willem Norde, Truus Posthuma-Trumpie
(Wageningen UR FBR)
http://www.bastiaanse-communication.com/html/rme2011_new.html
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