Scienion Workshop, Berlin, 21/22-10-2010

Antibody microarrays visualized by

carbon nanoparticles - from R to D

Aart van Amerongen

Agrotechnology & Food Sciences Group
 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 from
primary production up to end-products
 > 70% of overall budget is result of acquisition of projects
 'Customers': SMEs, multinationals, governments, EU, ......
 Business units:
 Food, Freshness and Chains
 Biobased Products
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
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 their
sensitivity, specificity, and cross-reactivity, the lack of
integrated systems that include fluid handling, sample
preparation and signal processing and inacceptable
background signals

 To apply protein multi-analyte assays many problems

have still to be overcome to end up with validated in vitro
diagnostics as indicated by Hartmann et al. (2009)
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

Multi-analyte diagnostics
 Binding of proteins to solid phase materials
 Protein microarrays:
 Proteins consist of over 20 different building blocks (amino
acids) 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 phase
materials
 From a functional point of view the adsorption of proteins
onto surfaces, membranes, or nanoparticles is even more
crucial

Arnoldus W.P. Vermeer, Willem Norde, Aart van Amerongen

(2000) The unfolding / denaturation of IgG of isotype 2b and its
FAB and FC fragments. Biophysical Journal 79, 2150-2154
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 on
solvent 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 its
FAB and FC fragments. Biophysical Journal 79, 2150-2154
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 the
individual proteins will not be optimal

physical or chemical bond IgG1 model; electric potential plot

Multi-analyte diagnostics
 Binding of proteins to solid phase materials
 Two-Compartment Model (TCM)
 Originally proposed for the analysis of mass-transport limited
biomolecular interactions in the Biacore instruments
 Compartments:
1. Transport of the analyte from the bulk
compartment to the surface reaction area
2. Subsequent binding process in reaction
compartment

Analytes Compartment 1

Binding molecules Compartment 2

Multi-analyte diagnostics
 Binding of proteins to solid phase materials
 TCM was modified for the analysis of interactions in the
microarray format by Kusnezow et al.*

 Conclusion:
Strong limitation by mass transport to the surface

 Kinetics may be slowed down as much as hundreds of

times compared to the solution kinetics
 Very important to address this point (e.g., by shaking and/or
increased incubation temperature)

*:
see, e.g., J.Chem.Phys. (2005); Proteomics (2006);
Mol.Cell.Proteomics (2006); Expert Rev.Mol.Diagn (2006)
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
Influence of various parameters on
the functionality of printed proteins
Non-contact inkjet printed proteins
STW project 10095: Biomolecules Substrate Topography
of Inkjet Printed Structures (Bio-STIPS)
Bio-STIPS
 Background:
 In many evaporation processes of practical importance such as
the evaporation of solvents filled with polymer or in DNA and
protein microarrays, the viscosity of the fluid increases
substantially with solute concentration, and hence during the
evaporation process

 This has a large influence on the shape of the deposition and

the distribution of (bio)molecules on the substrate, while the
functioning of e.g. the microarrays is critically dependent on this
distribution
Bio-STIPS participants
 Scientific collaborators:
 Eindhoven University of Technology, Dept. of Mechanical
Engineering (coordinator dr. Hans Kuerten)
 Wageningen University, Laboratory of Physical Chemistry and
Colloid Science (Prof.dr. Willem Norde)
 Wageningen UR Food & Biobased Research, Biomolecular
Sensing & Diagnostics (dr. Aart van Amerongen)
 User committee:
 Philips Research, Océ Technologies NV, Chematronics B.V.,
Animal Health Service Deventer, PamGene International B.V.,
Scienion AG, Eindhoven University of Technology (Mesoscopic
Transport Phenomena), Friedrich-Schiller-Universität Jena
(Lab. Organic & Macromolecular Chemistry)
Bio-STIPS
 Theoretical science (Eindhoven University of Technology)
 Development of an extended physical model for the calculation
of the thickness of deposition that results after evaporation of a
solvent from a droplet and of the distribution of (bio)molecules
on/in the substrate
• 'Extended' means that all physical phenomena that play an
important role in practical applications will be taken into account

 Subsequently, this physical model will be implemented in a

numerical computer program
Bio-STIPS
 Theoretical science (Eindhoven University of Technology)
 The complexity of the physical phenomena involved, in
particular the interaction between the solute molecules and the
substrate, makes experimental validation a necessity

 Moreover, relevant physical parameters, such as

(concentration-dependent) viscosity, diffusivity, evaporation
velocity and porosity of the substrate, are needed for the
development of the model
Bio-STIPS
 Experimental science (Wageningen University)
 Experimental techniques on evaporating droplets
 The influence of the substrate surface and buffer composition
on the orientation and conformation and, consequently, the
functionality of biomolecules is of crucial importance
 Model proteins will be deposited and the orientation and/or
conformation of these proteins will be studied by applying
surface 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 affinity
binding in the layer
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 µm
 Humidity-controlled
3 mm
Influence of various parameters on
the functionality of printed proteins
Buffer composition (and Humidity)
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 nanoparticles

with neutravidin after printing in: PBS ~70% CB ~70%

 Better intensities after printing

BSA-biotin in carbonate buffer
Reflectometry experiments with BSA-biotin
 Reflectometry is an optical technique for the determination
of the adsorption of molecules from solution onto a
macroscopically flat substrate

S
Qf mg/m2
S0
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 if

dissolved in PBS buffer
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:

Atomic Force Microscopy of BSA-biotin spots
 BSA in PBS (250 µg/mL; 1 droplet)
 Expected: smooth spot, uniformly distributed BSA-biotin
molecules
AFM pictures

Printed droplet Fluid evaporation Biomolecules on Biomolecules on

surface surface following first
wash / incubation
Atomic Force Microscopy of BSA-biotin spots
 BSA in PBS (250 µg/mL; 1 droplet)
 Observed:

 Height of structures: up to 1000 nm

• Monolayer of BSA would be ± 10 nm
Specific staining with fluorescent streptavidin
 Confocal Laser Scanning Microscopy

 No signal in areas where structures were removed during

blocking/washing step => even no monolayer of BSA !?
Influence of immobilization conditions
 Model – BSA-biotin (250µg/mL)
 Substrate: HTA polystyrene slide
 Spotting buffer : Carbonate buffer (pH 9.6)
 Average height of ~150nm

3D view Height data

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 ~ 400 nm
(pH 7.4)

CB ~ 240 nm
(pH 9.6)
Influence of immobilization conditions
 Omitting NaCl from PBS resulted in better / good spots
 Imaging method: Tapping mode
 Substrate: HTA polystyrene slide
PBS PB CB
(pH 7.4) (pH 7.4) (pH 9.6)

BSA-bt
200µg/mL

IgG-bt
200µg/mL
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

~22% [BSA-bt]=250µg/mL (2 drops)

~70% [BSA-bt]=250µg/mL (2 drops)

6e+5 6e+5

Z Data
Z Data

0 4e+5 0
4e+5
2e+5 2e+5
4e+5 4e+5
6e+5 6e+5
20 2e+5 20
2e+5 18 18
16 16
14 14
12 12

ta
ta

Da
10
Da

0 10 0
8 18 16 8

X
18 16
X

14 6 14 6
12 12 4
10 4 10
8 8
6 2 6 2
Y Da 4 Y Da 4
ta 2 ta 2
Influence of various parameters on
the functionality of printed proteins
Substrate hydrophobicity
Protein binding to substrates
 For the protein in aqueous solution, the hydrophilic side
chains are presented at the outside and the hydrophobic to
the inner of the protein to avoid contact with water
 Attached to a surface, new interactions become possible
for the protein; in the first step, the protein rearranges its
structure to reach an energetically advantageous
conformation
 Because of interactions with adjacent protein molecules
and the surface, additional rearrangements can occur,
resulting in a partly or totally denaturated protein
 Are these adsorbed protein molecules still functional ?

Christine Müller, Anne Lüders, 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) 4136–4141
Protein binding to substrates
 Adsorption model:

From: Mondon M.Untersuchungen zur Proteinadsorption

aufmedizinisch relevanten Oberflächen mit Rasterkraft-
spektroskopie und dynamischer Kontaktwinkelanalyse.
Ph.D. Thesis, University of Kaiserslautern, 2002
Substrate hydrophobicity
 Substrate hydrophobicity influences
the deposition of protein molecules

 On hydrophilic surfaces (A)

droplets spread
 Spreading of a droplet induces
internal advection and
enrichment of dissolved protein
molecules at the droplet
perimeter ('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, György Marko-Varga and Thomas Laurell. Porous
silicon protein microarray technology and ultra-/superhydrophobic
states for improved bioanalytical readout. Biotechnology Annual
Review 13 (2007)
 Contact angle of water droplet on silanized glass
Contact
Material Silane derivative 2 µL droplet in Goniometer
angle

Glass < 10°

3-Cyano Propyl Triethoxy

CPTES ~ 49°
Silane

3-Glycidyloxy Propyl
GPTMS ~ 61°
Trimethoxy Silane

PhECS Phenyl Ethyl Chloro Silane ~ 75°

HMDS Hexa Methyl DiSilazane ~ 88°

DCDMS DiChloro Dimethyl Silane ~ 102°

Substrate hydrophobicity
 Influence on protein distribution
 Printed: IgG molecule with biotin attached
 Specific staining by streptavidin with attached
fluorochrome Alexa-633
 Confocal Laser Scanning Microscopy

Untreated GPTMS DCDMS

< 10° ~ 61° ~ 102°
CLSM
Alexa-633-
Streptavidin
Increase of surface area to
volume ratio

Nitrocellulose substrate and Carbon nanoparticles

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 by
decreasing spot diameter
 Examples with carbon
nanoparticles
 Verotoxigenic Escherichia
Nexterion
coli O157
 Malaria species detection
Colloidal carbon nanoparticles

Introduction to carbon nanoparticles

Carbon nanoparticles
 Characteristics:
 Clusters of primary particles => 50 - 400 nm, depending on the
carbon pigment used, with or without surfactants / detergents
 Advantage in terms of sensitivity and Hook-effect

---- 1 µm

----- 10 µm

----- 100 nm
Sensitivity of lateral flow labels: Julian Gordon and Gerd Michel; Foundation
for Innovative New Diagnostics (FIND)

Carbon nanoparticles in top-10; low picomolar detection

Analytical sensitivity limits for lateral flow immunoassays. Clinical

Chemistry 54 (2008) 1250-1251. Including Table 1 + Supplement
FindDiagnosticsOrg (www.clinchem.org/cgi/data/clinchem.2007.102491/DC1/1)
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 => <= NALFIA / NAMIA

Nucleic Acid LFIA (NALFIA) or MIA (NAMIA)
 Rapid detection of genetic material
 Examples: micro-organisms (human, veterinary, feed / food
pathogens)
 Procedure (PCR procedure down to 15 minutes):

tag 1 primer 1

amplification with double-strand

template DNA double-tagged
(e.g. PCR protocol) amplicon
primer 2 tag 2

 Alternative procedure: amplification to single chain

amplicons that are hybridized to tag-labelled probes
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 with
neutravidin
 Antibodies spotted onto the membrane
recognize one of the tags
 Specificity is in the combination of
forward primer and tag
• Any target can be assigned to a particular
line (NALFIA) or spot (NAMIA)
Examples of using carbon nanoparticles
VTEC diagnostics:
Rapid, multi-analyte assays for the detection of genes coding
for verotoxigenic Escherichia coli O157 (VTEC) and four
virulence factors
Collaboration in the context of the Diagnostics Platform of Wageningen UR
(A. de Boer, K. Maassen, F.J. van der Wal)
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
Verotoxigenic E. coli (VTEC)
 Virulence factors:
 Elaborate phage-encoded cytotoxins: verotoxins
• vt1
• vt2
 Intimin protein (VTEC attachement to intestine epithelial cells)
• eae
 Enterohaemolysin
• ehec
VTEC research goals
 Development of two rapid molecular biological methods
with carbon nanoparticles as signal labels to detect the
presence 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)
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' biotin
vtx2b vt2-F Verotoxin 2 5'-GGGCAGTTATTTTGCTGTGGA-3' 130 bp FITC
Vt2-R 5'-GAAAGTATTTGTTGCCGTATTAACGA-3' biotin
eae eae-F Intimin 5'-CATTGATCAGGATTTTTCTGGTGATA-3’ 102 bp DIG
eae-R adhesion 5'-CTCATGCGGAAATAGCCGTTA-3’ biotin
ehxA ehec-F Hemolysin 5'-CGTTAAGGAACAGGAGGTGTCAGTA-3' 142 bp DMP
ehec-R 5'-ATCATGTTTTCCGCCAATGAG-3' biotin
16S Hui -F Small ribosome 5'-CATGCCGCGTGTATGAAGAA-3' 96 bp Cy5
hui-R subunit 5'-CGGGTAACGTCAATGAGCAAA-3' biotin

PCR reaction (for all primers)

PCR program Reagents
Temp time Water 5 µL
98 ºC 30 s Phire Buffer 5x 4 µL final [MgCl2] = 1,5 mM
98 ºC 5s ┐ dNTP 5 µL final [dNTP] = 0,25 µM
61 ºC 5s ├30 cycles Primers (10 µM) 1,8 µL final [primer] = 0,9 µM (each)
72 ºC 5s ┘ Phire polymerase 0,4 µL
72 ºC 1m Template 2 µL
4ºC ∞ Total volume 20 µL
30 min
Results of VTEC NALFIA and NAMIA
 Nucleic acid-based detection of verotoxigenic E.coli O157
and 4 virulence factors; trial with 48 field-derived E.coli
samples

 NALFIA: Line/Gene Antibody mg/mL control

control IgG-biotin 200 α-TXR (vt1)
α-FITC (vt2)
vt1 Texas Res 200 α-DNP (ehec)
vt2 FITC 50 α-DIG (eae)
α-Cy5 (16S)
ehec DNP 200
eae DIG 200
16S Cy5 800 B vt1 vt2 ehec eae 16S all
Results of VTEC NALFIA and NAMIA
 Nucleic acid-based detection of verotoxigenic E.coli O157
and 4 virulence factors; trial with 48 field-derived E.coli
samples

 NAMIA: vt1

vt2

16S

eae

ehec

control

blank

 Average pixel grey levels were automatically calculated and

scored positive if > 3 x background SD
 VTEC NALFIA and NAMIA compared to Q-PCR
 Comparison:

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

• NALFIA manuscript accepted by Analytical and

Bioanalytical Chemistry, in press
• Microarray manuscript in preparation
Examples of using carbon nanoparticles
Malaria diagnostics:
Species identification and SNPs analysis related to
artemisinin drug combination therapy resistance
Malaria diagnostics
 Multi-drug resistance in malaria under combination
therapy: Assessment of specific markers and development
of innovative, rapid and simple diagnostics
 Project acronym: MALACTRES
 EU7 - HEALTH project
 2008 - 2012
 Participants: Beneficiary
Number
Beneficiary name Beneficiary
short name
Country

1 Royal Tropical Institute KIT The Netherlands

(coordinator)
2 Institute of Tropical Medicine ITM Belgium
3 London School of Hygiene and Tropical LSHTM United Kingdom
Medicine
4 Biomolecular Sensing & Diagnostics, AFI The Netherlands
Wageningen University and Research Centre
5 Forsite Diagnostics Limited FDL United Kingdom
6 Tropical Diseases Research Group, Faculty of UNIBEN Nigeria
Pharmacy, University of Benin
7 Centre Muraz CM Burkina Faso
8 Kilimanjaro Christian Medical Centre KCMC Tanzania
Malaria diagnostics
 Facilities in Africa (Burkina Faso)
 Research institute in Bobo Dioulasso

 Hospital near Ouagadougou

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 Plasmodium species

 Trial in Kenya
Malaria diagnostics - NALFIA
 NALFIA compared with microscopy (gold standard) and
agarose gel electrophoresis
 650 clinically suspected malaria
cases
 NALFIA detection limit: 0.3 - 3
parasites per µL
 More sensitive than microscopy
 10-fold more sensitive than gel
electrophoresis
 Excellent agreement with gel
electrophoresis

Petra F. Mens, Aart van Amerongen, Patrick Sawa, Piet A. Kager, Henk
D.F.H. Schallig. Molecular diagnosis of malaria in the field: development of a
novel 1-step nucleic acid lateral flow immunoassay for the detection of all 4
human Plasmodium spp. and its evaluation in Mbita, Kenya. Diagnostic
Microbiology and Infectious Disease 61 (2008) 421-427
Malaria diagnostics - NALFIA
 Recent trial in Burkina Faso
 Four-lines NALFIA (GAPDH,
Plasmodium falciparum, Plasmodium
vivax, Pan-Plasmodium)

 Initial statistics:
 Plasmodium falciparum and Pan-
Plasmodium excellent sensitivity
(97 and 98%, respectively)
Examples of the 4-lines PCR-
NALFIA with a P.falciparum (left)
 Plasmodium vivax sensitivity only
and a P.vivax specific sample 57% (but: Pan-Plasmodium OK)
Malaria diagnostics - Microarray method
 Anti-tag antibodies printed on Nexterion slides:
• 128, 320 and 780 ng/μl anti-TexasRed
• 32, 80 and 200 ng/μl anti-Dig
• 16, 40 and 100 ng/μl anti-FITC
• 64, 160 and 380 ng/μl anti-DNP

Microtiter plate-sized holder for 4

slides; 64 samples
Malaria diagnostics - Microarray method
 Added reagents (one-step incubation; 1 h)
 PCR (multiplex): 4 µL
 Carbon conjugate: 5 µL
• Neutravidin - Alkaline phosphatase (AP) fusion protein

 Following incubation the lower half

of the slide was incubated with AP
substrate (10 min incubation)
 Precipitating dye increases signal
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)
MicroVigene microarray analysis software
 Following digitization of the slides (16 microarrays) by
flatbed scanning or digital camera the software
automatically 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
Malaria diagnostics - Microarray method
 Single-laboratory trial
 Microarray design
• Spots available for other
targets like SNPs

 Procedure: Carbon nanoparticles (1 hour) followed by 3 short

wash steps with running buffer, alkaline phosphatase substrate
(10 min) and a short wash step with MQ
 Malaria diagnostics - Microarray method
 Nexterion slide; various combinations of amplicons

Amplicons: GAPDH GAPDH GAPDH GAPDH PCR without

template
falc falc falc
vivax vivax vivax
Pan Pan Pan Pan

Concentration of individual amplicons: 0.8 pmol Concentration Concentration of Of all four

of amplicons in amplicons in PCR
combination: combinations: 0.3 pmol reactions:
0.2 pmol 1 µL

 Microarray trial in Africa scheduled in spring 2011

New assay formats with carbon
nanoparticles
 Microarrays in wells of ELISA plates
 Lateral flow microarray immunoassay (LMIA)
New assay formats with carbon labelling
 Microarrays in wells of microtiter plates
 As compared to conventional ELISA: multi-
analyte assay with the same sample
volume
 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)
New assay formats with carbon labelling
 Lateral flow Microarray ImmunoAssay (LMIA)
 Speed of LFIA (5 to 10 minutes)
• An even better surface area to volume ratio
as compared to microarrays with incubation
from samples on top
 Multi-analyte level of microarray <= impression

 Ease of handling like LFIA

 Preparation of microarrays on nitrocellulose
membranes in dedicated holder on vacuum
plate
 Digitization and quantification possible
• CCD camera, flatbed scanning
Concluding remarks
 Carbon nanoparticles are excellent labels for rapid lateral
flow and microarray methods

 The signal-to-noise ratio of these nanoparticles enables

low picomolar detection by visual inspection

 Digitization and quantification of the results can be

automated
Rapid Methods Europe 7: 24-26 January 2011, Noordwijkerhout, The Netherlands
http://www.bastiaanse-communication.com/html/rme2011_new.html

Thank you for your attention !

Acknowledgements:
 René 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)

aart.vanamerongen@wur.nl © Wageningen UR