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Protein discovery and development of protein therapeutics require assays that are highly sensitive. The sensitivity of a bioassay is fundamentally limited by the noise in an assay system

with non-specific binding being the most prevalent one. Here, we developed a sandwich assay on an antibody nanoarray. By counting the dots with positive fluorescence signal or scattered

light emitted by silver particles1, we inferred the protein concentrate in a sample. We quantified 14 proteins that have a general relevance to caner and that are believed to play a role in

cancer development thus the detection of these proteins at low abundance may lead to early disease diagnosis and better prognosis.

Introduc)on  

Digital  Assay  with  noise  rejec)on   Imaging  Analysis  

Image analysis and signal quantification. a) Original image. b) Gaussian blur of the original image. c) Image after background subtraction and normalization. d) Rotated imaged calculated using Radon transformation. e)-f) The overlay of a template with the normalized, rotated image for both foreground and background extraction. g) Histogram of the foreground and background signals.

Conclusion  

1Department  of  Biomedical  Engineering,  McGill  University,  Montreal,  QC  Canada  2Department  of  Chemistry,  McGill  university,  Montreal,  QC  Canada    

Gina  Zhou1,  Amani  Hariri2,  Gonzalo  Cosa2,  David  Juncker1  

 A  Digital  Assay  on  a  Nanoarray  with  Noise  Rejec)on  

Binding  curves  and  comparison  with  fluorescence  microarray  assays  

References  

•  Nanoarray patterned binding surface provides noise rejection of non-specific binding signals in an assay by spatial discrimination.

•  Singleplex fluorescence digital assay reaches a LOD of 30 ag/mL LOD and multiplexed silver-enhanced digital assay an LOD of 10 fg/mL – 10 pg/mL LOD, both with single molecule resolution.

1. Protein targets in a 16-plex assay •  Growth factor/receptor: bNGF, EGF, ENDO, Her2, •  Interleukins: IL1b, IL3, IL8 •  Cytokine: GCSF, MCSF •  Proteins related to apoptosis: FAS, FAS-L, TNFa •  Proteins related to cancer metastasis:ANG1, EpCAM •  Two house keeping IgGs

Schematic of digital assay. (1) cAbs immobilized on a microarray slide by nanocontact printing2. The dots are 150 nm in diameter, spaced at 2 µm, constituting 5M dots over a surface area of 6x6 mm2. (2) A sandwich assay was performed on the nanoarray patterned surface. (3) Colocalized spots were counted as a hit signal and the %Hits was calculated to infer protein concentration.

Two-­‐color  TIRF  microscopy  

Fluorescence images of a digital assay. (a-b) Digital assay binding surface. (c) Colocal ized f luorescence image of fluorescence digital assay results. The circles highlight the missing spots and the arrows point to non-specific binding signals.

20 µm  

5 µm   5 µm  

Limit of detection (pg/mL) This assay

Fluorescence microarray assay3

ELISA (R&D4)

ANG1 0.03 25.23 10.3 bNGF 7.44 2.01* na EGF 0.42 56.5 0.7

ENDO 0.14 28.7* 30 EpCAM 2.23 3.86* na

FAS 3.27 185.4 20 FAS-L 4.44 407.7 8.1 GCSF na 71.5 20 HER2 0.75 1132.4 na IL1b 0.82 12.3 1 IL3 0.04 20.49* 7.4 IL8 na 11.3 na

M-CSF 0.01 422.4 9 TNFa 0.03 15.6 5.5

Binding curves of Il1b obtained by singleplex fluorescence digital assay(blue), multiplex silver-enhanced digital assay (green) and and fluorescence microarray assay(red). The LODs were estimated to be 32 ag/mL, 0.82 pg/mL, and 12 pg/mL, respectively.

3. LOD Comparison of digital assay and other assays

1.  Zhou, G. et al. IEEE Transducers, 2783-86 (2013)

2.  Ricoult, S. G. et al. Small 9, 3308-3313 (2013)

3.  Pla-Roca, M. et al. Mol Cell Proteomics 11 (2012)

4.  R&D Systems. ELISA reference guide and catalog

Photobleaching experiment to validate single binding on nanodots. (a) Green traces show the photobleaching of one or two dyes on a a single nanodot; red traces are the noise floor. The numbers of “steps” indicates the number of dyes per dot. (b) At less than 0.2% active binding surface, ca. 90% of nanodots has a single molecule.

a  

b  

2. Binding curves of 12 proteins

Quan)fica)on  of  IL1b    

Valida)on  of  single  molecule  binding  

*: unpublished data ConcentraAon  [pg/mL]  

%Hit