Multiplexed Digital Assays With Automated Image Acquisition And Analysis Gina Zhou1*, Derin Sevenler2, Jacob Trueb2, Selim Unlu2, Andy Ng1 and David Juncker1

1Biomedical Engineering, McGill University, Canada and 2Electrical and Computer Engineering, Boston University, USA

Introduction

Multiplexed Digital Assay

Digital Assay

Conclusion

Disease diagnosis can benefit from highly-sensitive multiplex assays to simultaneous quantification of analytes and biomarkers. Sandwich assays are the most sensitive and specific format, and have recently been developed as digital assays where concentration is not measured as an average signal, but as a number of binding events that can be recorded with up to single molecule sensitivity. Digital assays outperform classical sandwich assays such as ELISA by 3-4 orders of magnitude and can reach limits of detection (LOD) down to aM concentrations.1 Efforts have been made to multiplex digital assays, but have remained limited. One study detected single binding events on microarrays, but labelled the entire sample and used a direct assay.2 Multiplexing with sandwich assays was also shown, but only up to 4-plex.3

Here, we introduce a multiplexed digital assay (MDA) using a nanoarray-in-microarray format, enzymatic silver amplification,4 an automated image acquisition system, and an automated image acquisition and signal processing algorithm. These results constitute the first multiplex digital sandwich assay on a microarray for 12 analytes, with LOD in the sub-fM range, and an excellent dynamic range when compared with conventional fluorescence microarray assays.

References 1.  Kim, S. H. et al. Large-scale femtoliter droplet array for digital counting of single biomolecules. Lab Chip 12, 4986-4991 (2012) 2.  Schmidt, R. et al. Single-molecule detection on a protein-array assay platform for the exposure of a tuberculosis antigen. J of Prot Res 10, 1316-1322

(2011) 3.  Rissin, D. M. et al. Multiplexed single molecule immunoassays. Nat Biotech 28, 595-599 (2010) 4.  Zhou, G. et al. High-Performance low-cost antibody microarrays using enzyme-mediated silver amplification. J. of Prot Res 14, 1872-1879 (2015) 5.  Ricoult, S. G. et al. Large dynamic range digital nanodot gradients of biomolecules made by low-cost nanocontact printing for cell haptotaxis. Small 9,

3308-3313 (2013) 6.  Pla-Roca, M. et al. Antibody Colocalization Microarray: A scalable technology for multiplex protein analysis in complex samples. Mol. Cell. Proteomics 11

(2012)

•  A MDA platform was developed using low-cost and high throughput nanocontact printing and inkjet microspotting, and automated image acquisition and analysis.

•  A 12-plex MDA assay with single particle resolution achieved sub-fM limits of detection for the best antibodies, outperforming fluorescence microarrays.

•  Large-scale multiplexing could be accomplished by an additional dAb spotting, to colocalize6 cAb and dAb and overcome reagent-driven cross-reactivity. Acknowledgements

Process flow of a multiplexed digital assay. (a) Anti-mouse IgGs immobilized on a microarray slide by nanocontact printing. (b) Surface is blocked, and (c) 12 cAbs inkjet printed as microarray on the nanopattern. (d) Silver enhancement performed and (e) assay result imaged with an automated microscope stage. (f) Dark field image of silver precipitation signals on a nanodot array. (g)-(i) SEM images of a nanoarray at different magnification, confirming that only one silver particle is found on a single nanodot.

Automated Image Acquisition Schematic of the image acquisition process and setup. (a) Data flow of the image acquisition system, consisting of a darkfield microscope, an automated 3-axial stage, communication units, and user interface with image analysis software. (b) Schematic of the darkfield optics, with arrows showing the light path. (c) The complete imaging setup in the laboratory.

Image Processing, Signal Extraction, and Data Analysis Algorithm Image analysis and signal quantification for MDA. (a) Raw image data. (b) Gaussian blurred image highlighting the varying global background of the raw image. (c) Normalized image after background subtraction. (d) Image is rotated and (e) the nanopattern aligned for signal extraction. (f) Histogram of log-transformed signal intensity, red asterisk indicates the determined signal threshold.

12-plex Multiplexed Digital Assay Results Optimization of microarray spot size and an excerpt of a 12-plex MDA. (a) Scaling law of microspot diameter vs the number of plex and digital assay dynamic range. (b) 9 darkf ie ld images stitched, showing microspots of ErbB2/Her2, Fas ligand, and IL-1b on a nanopattern. (c) Darkf ie ld image of nanodot patterns within the boundary of a microspot. (d) Binding curve of IL-1B obtained by MDA and by a conventional fluoresence m i c r o a r r a y a s s a y. ( e ) Comparison of LOD and assay range obtained by this M D A a n d b y E L I S A s (provided by vendors), as well as the known serum level of the measured 12 proteins.

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

a b c

e

7 fg/mL

1.3 pg/mL

Sandwich Immunoassay with Silver Amplification Schematic of a sandwich immunoassay with silver amplification4. Capture antibody (cAb) and biotin-labeled detection antibody (Biotin-dAb) mutually binds a protein. Horse raddish peroxidase conjugated-streptavidin then binds Biotin-dAb, precipitates silver ions and generates a binding signal.

Y

Y Ag

+Ag

YYcAbBiotin-dAbProteinSA-HRPAg