Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Summary

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

Abstract

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that <40 μm alignment has been achieved with double transfer, reaching an array density of 625 spots/cm². A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

Introduction

A panel of biomarkers comprising multiple proteins may provide higher sensitivity and specificity than a single biomarker in the diagnosis of complex diseases such as cancers^1,2. The enzyme-linked immunosorbent assay (ELISA) has been the gold standard technology used in clinical laboratories achieving a limit of detection at low pg/mL in plasma, but limits to one target per assay^3,4,5. Antibody microarrays have been developed for accommodating thousands of miniaturized assays conducted in parallel on a single microsco....

Protocol

1. Fabrication and storage of snap chips

1. Single transfer method (Figure 1a)
   1. Spot cAb solutions containing 400 µg/mL antibodies and 20% glycerol in phosphate-buffered saline (PBS) onto a nitrocellulose (or a functionalized glass) assay slide with an inkjet microarray spotter¹³ at a relative humidity of 60% (1.2 nL for each spot) with 800 µm center-to-center spacing. Make sure the slide is fixed on the spotter deck according to one corner (here, the bottom left corner was used).
   2. Incubate the spotted assay slide at room temperature for 1 h with a relative humidity of 60%.

Results

The assay procedure for both single and double transfer methods is shown in Figure 1. In single transfer, the cAbs are spotted directly on the assay slide and the dAbs are transferred onto the assay slide upon use in a mirror pattern of the cAbs (Figure 1a). Only one transfer procedure is required, but this method suffers from misalignment between the two microarrays, mainly caused by the angular misalignment between the slide an.......

Discussion

In this work, we have presented a snap chip technology that makes the cross-reactivity-free multiplex immunoassays widely available for the researchers with basic experimental setup. Different from existing antibody microarrays, no microarray spotter is needed for end-users. Both single and double transfer methods are demonstrated, and double transfer affords superior alignment accuracy down to ~ 40 μm for 98% spots, with the largest misalignment of 63 µm¹⁴. A novel snap apparatus was de.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phosphate buffered saline tablet	Fisher Scientific	5246501EA
Streptavidin-conjugated Cy5	Rockland	s000-06
Tween-20	Sigma-Aldrich	p1379
Bovine serum albumin	Jackson ImmunoResearch Laboratories, Inc	001-000-162
Glycerol	Sigma-Aldrich	G5516
Blocking solution: BSA-free StabilGuard Choice Microarray Stabilizer	SurModics, Inc	SG02
Nitrocellulose coated slides	Grace Bio-Laboratories, Inc	305116
Aminosilane coated slides	Schott North America	1064875
Snap Device	Parallex BioAssays Inc.	PBA-SD01
Inkjet microarray spotter	GeSiM	Nanoplotter 2.0
Slide module gasket	Grace Bio-Laboratories, Inc	204862
Humidity Stabilization Beads	Parallex BioAssays Inc.	PBA-HU60
Array-Pro Analyzer software	Media Cybernetics	Version 4.5
Fluorescence microarray scanner	Agilent	SureScan Microarray Scanner
Biostatistics software	GraphPad Software	GraphPad Prism 6
Endoglin capture antibody	R&D Systems	MAB10972
Endoglin protein	R&D Systems	1097-EN
Endoglin detection antibody	R&D Systems	BAF1097
IL-6a (see Table 1)	R&D Systems
IL-6b (see Table 1)	Invitrogen