Antibody microarrays visualized by carbon nanoparticles -from R to D Aart van Amerongen University...

8/8/2019 Antibody microarrays visualized by carbon nanoparticles -from R to D Aart van Amerongen University Wageningen

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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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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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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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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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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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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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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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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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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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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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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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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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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


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

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10

12

14

161820

24

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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

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10

12

14

161820

24

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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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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 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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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
http://www.finnzymes.com/movies/loading.html


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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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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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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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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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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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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:


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

Antibody microarrays visualized by carbon nanoparticles -from R to D Aart van Amerongen University...

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Transcript of Antibody microarrays visualized by carbon nanoparticles -from R to D Aart van Amerongen University...