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 microscope slide^6,7,8. However, the multiplexing capability of this method is limited by reagent-driven cross-reactivity, arising from the application of a mixture of dAbs, and it becomes more problematic with an increasing number of targets^9,10,11. Pla et al. have stated that the resulting vulnerability of a multiplex sandwich assay scales as 4N(N-1) where N is the number of the targets¹².

To mitigate cross-reactivity in antibody microarrays, antibody colocalization microarray (ACM) has been developed in our laboratory for multiplex sandwich assay¹². Capture antibodies (cAbs) are spotted on a substrate with a microarray spotter. After blocking samples are applied on the surface, and then individual dAbs are spotted on the same spots with the cAb-antigen complex. All cross-reactivity scenarios between antibodies and antigens can be mitigated with ACM, and limits of detection at pg/mL have been achieved. However, the assay protocol requires preparing and spotting the dAbs during the experiments using an on-site microarray spotter with high precision for alignment purpose, which is expensive and time consuming, limiting the wide application of this technology in other laboratories. A handheld ACM, named snap chip has been developed for cross-reactivity-free and spotter-free multiplex sandwich immunoassays^13,14,15. cAbs and dAbs are pre-spotted onto an assay slide and a transfer slide respectively in microarray format and stored. During the assay, the slides are retrieved and a microarray of dAbs are transferred collectively onto the assay slide by simply snapping the two chips together. A snap apparatus is used for reliable reagent transfer. Nitrocellulose coated slides with a relatively large antibody binding capacity have been used as the assay slides to absorb the liquid droplets and thus facilitating reagent transfer, however, the slides are more expensive than regular glass slides and microarray scanners compatible with non-transparent slides are needed for signal acquisition.

In this work, we demonstrate the protocol of performing a multiplex sandwich immunoassay with a snap chip. A novel snap apparatus has been developed for more convenient and reliable reagent transfer from microarray-to-microarray. Importantly, here we have established the reagent transfer method onto regular glass slides with the snap chip. 1024 spots were successfully transferred and aligned onto a glass slide, significantly expanding the use of this technology in most laboratories.

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%.
   3. Clamp a slide module gasket with 16 compartments on the assay slide to divide it into 16 wells. Rinse the slide three times by adding 80 µL of PBS containing 0.1% Tween-20 (PBST) into each well, and shaking at 450 rpm on a shaker, 5 min each time.
   4. Add 80 µL of blocking solutions to each well and shake for 1 h at 450 rpm.
   5. Remove the gasket and dry the assay slide with a stream of nitrogen.
   6. Fix a transfer slide on the spotter deck and push the bottom left corner of the slide against bottom left corner of the spotter deck. Inkjet spot an array of alignment marks (a solution of polystyrene micro-beads) with the same layout as that for the cAbs.
   7. Let the alignment marks dry. Flip the transfer slide and put it back onto the spotter deck, fixing against the bottom left corner.
   8. Prepare the dAb spotting solutions containing 20 µg/mL antibodies, 20% glycerol, and 1% BSA.
   9. Using the spotter's camera, take a picture of an alignment mark. Implement this picture into the spotting program as a fiducial and get the image recognition system of the inkjet spotter to identify the most top left mark. Use its coordinate as the first spot of the dAb array. Spot 8 nL per droplet with a center-to-center spacing of 800 µm.
2. Double transfer method (Figure 1b)
   1. Place a transfer slide 1 on the inkjet spotter deck against the bottom left corner. Spot cAbs solutions containing 400 µg/mL antibodies, 20% glycerol, and 1% BSA in PBS onto the slide with an inkjet microarray spotter at a relative humidity of 60% (0.4 nL for each spot and ~ 200 µm in diameter). The center-to-center spacing is 400 µm.
   2. Snap the transfer slide with a nitrocellulose coated (or a functionalized glass) assay slide using a snap apparatus (Figure 2) to transfer the cAb droplets onto the assay slide.
      1. Turn all six buttons on the side of the snap apparatus by 90 degrees to pull and lock all the plungers in the open position. Insert the transfer slide into the slide holder in its receptacle with the clipped corner facing the fixed pogo pin. Turn the first set of three buttons to release the plungers with the golden pogo pin and make sure the slides are pushed against the alignment pins.
      2. Insert a nitrocellulose-coated slide in the snap apparatus upside-down sitting on the 4 pogo pins. Turn the second set of three buttons to release plungers with the silver pin and make sure the slides are pushed against the alignment pins.
      3. Close the snap apparatus by placing the top shell on the slide holder using the alignment pillars and holes for a precise positioning.
      4. Insert the closed snap apparatus into its cage and push the closure tab completely down to bring the microarrays face-to-face while applying the appropriate pressure. Keep closed for 1 min.
   3. Separate the slides. Incubate the assay slide at room temperature for 1 h with a relative humidity of 60%. Wash, block, and dry the assay slide as described in steps 1.1.3 - 1.1.5.
   4. Place another transfer slide on the inkjet spotter deck and push against the bottom left corner. Spot dAb solutions containing 50 or 100 µg/mL of antibodies (see Table 1), 20% glycerol, and 1% BSA in PBS. Ensure that each spot is 0.8 nL and the center-to-center spacing between spots is 400 µm.
   5. Store the assay and the transfer slide. Seal both assay and transfer slides in an airtight bag containing desiccant and put in a -20 °C freezer.

2. Multiplexed immunoassays with snap chips

1. Retrieve the assay slide from the freezer. Leave the bag sealed for 30 min until the slide comes to room temperature.
2. Prepare 7-point serial diluted sample solutions by spiking proteins in PBS containing 0.05% Tween-20.
   NOTE: Here, a 5-fold dilution factor is used. The starting concentrations for each protein are listed in Table 1.
3. Prepare sample solutions as appropriate. For example, dilute human serum 4 times in PBST buffer.
4. Clamp a 16-compartment gasket on the assay slide.
5. Fill one column of 8 wells with the 7 protein dilution solutions and a protein-free PBST buffer solution by pipetting. Pipette the sample solutions in the other 8 wells on the same slide.
   NOTE: 80 µL solutions can be fit in each well. More slides can be used to measure additional samples when necessary.
6. Incubate the samples on a shaker at 450 rpm either for 1 h at room temperature or for overnight at 4 °C. Wash the slide three times with PBST on the shaker at 450 rpm, 5 min each time. Remove the gasket and dry the slide under a stream of nitrogen gas.
7. Retrieve the transfer slide with dAbs from the freezer. Keep the bag sealed for 30 min at room temperature. Then, incubate the slide in a closed chamber (e.g. empty tips box) containing 60% humidity stabilization beads for 20 min for rehydration.
8. Snap the transfer slide with the assay slide using a snap apparatus (Figure 2) to transfer the dAb droplets. See section 1.2.2 for the operation of the snap apparatus.
9. Separate the slides. Incubate the assay slide in a closed chamber containing 60% humidity stabilization beads for 1 h. Clamp the assay slide with a 16-compartment gasket and rinse the slide 4 times using PBST on a shaker at 450 rpm, 5 min each time.
10. Pipette 80 µL of solutions containing 2.5 µg/mL streptavidin fluorophore in PBS into each well. Incubate for 20 min on a shaker at 450 rpm.
11. Rinse the slide 3 times with PBST and once with distilled water on the shaker at 450 rpm. Remove the gasket and dry the slide using nitrogen gas.

3. Slide scanning and data analysis

1. Scan the assay slide with a fluorescence microarray scanner using the 635-nm-laser.
2. Extract the net intensity of each spot using an analysis software (e.g. array-pro analyzer)¹⁶.
3. Calculate the limit of detection (LOD) of each protein using a statistics software and determine the protein concentrations in the samples.
   NOTE: The LOD is defined as the Y-intercept of the standard curve incremented by three times the standard deviation of three independent assays.

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

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