Name: snap chip for cross-reactivity-free and spotter-free multiplexed sandwich immunoassays
Uploaded: 2017-11-13T12:30:00
Description: Watch this Scientific Journal Video about Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays at JoVE.com

We thank Dr. Rob Sladek for the use of the inkjet spotter. We acknowledge the final support from the Canadian Institutes for Health Research (CIHR), the Natural Science and Engineering Research Council of Canada (NSERC), the Canadian Cancers Society Research Institute and the Canada Foundation for Innovation (CFI). D.J. thanks support from a Canada Research Chair.

1. Fabrication and storage of snap chips
<ol>
	<li>Single transfer method (Figure 1a)
	<ol>
		<li>Spot cAb solutions containing 400 &#181;g/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 &#181;m center-to-center spacing. Make sure the slide is fixed on the spotter deck according to one corn...

<ol>
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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 &#956;m for 98% spots, with the largest misalignment of 63 &#181;m14. A novel snap apparatus was de...

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 cancers1,2. 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 assay3,4,5. Antibody microarrays have been developed for accommodating thousands of miniaturized assays conducted in parallel on a single microscope slide6,7,8. 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 targets9,10,11. 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 immunoassays13,14,15. 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.

<table>
	<tbody>
		<tr>
			<td>Phosphate buffered saline tablet</td>
			<td>Fisher Scientific</td>
			<td>5246501EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-conjugated Cy5</td>
			<td>Rockland</td>
			<td>s000-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>p1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Jackson ImmunoResearch Laboratories, Inc</td>
			<td>001-000-162</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking solution: BSA-free StabilGuard Choice Microarray Stabilizer</td>
			<td>SurModics, Inc</td>
			<td>SG02</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose coated slides</td>
			<td>Grace Bio-Laboratories, Inc</td>
			<td>305116</td>
			<td></td>
		</tr>
		<tr>
			<td>Aminosilane coated slides</td>
			<td>Schott North America</td>
			<td>1064875</td>
			<td></td>
		</tr>
		<tr>
			<td>Snap Device</td>
			<td>Parallex BioAssays Inc.</td>
			<td>PBA-SD01</td>
			<td></td>
		</tr>
		<tr>
			<td>Inkjet microarray spotter</td>
			<td>GeSiM</td>
			<td>Nanoplotter 2.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide module gasket</td>
			<td>Grace Bio-Laboratories, Inc</td>
			<td>204862</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidity Stabilization Beads</td>
			<td>Parallex BioAssays Inc.</td>
			<td>PBA-HU60</td>
			<td></td>
		</tr>
		<tr>
			<td>Array-Pro Analyzer software</td>
			<td>Media Cybernetics</td>
			<td>Version 4.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microarray scanner</td>
			<td>Agilent</td>
			<td>SureScan Microarray Scanner</td>
			<td></td>
		</tr>
		<tr>
			<td>Biostatistics software</td>
			<td>GraphPad Software</td>
			<td>GraphPad Prism 6</td>
			<td></td>
		</tr>
		<tr>
			<td>Endoglin capture antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB10972</td>
			<td></td>
		</tr>
		<tr>
			<td>Endoglin protein</td>
			<td>R&#38;D Systems</td>
			<td>1097-EN</td>
			<td></td>
		</tr>
		<tr>
			<td>Endoglin detection antibody</td>
			<td>R&#38;D Systems</td>
			<td>BAF1097</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-6a (see Table 1)</td>
			<td>R&#38;D Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IL-6b (see Table 1)</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

snap chip for cross-reactivity-free and spotter-free multiplexed sandwich immunoassays

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 &#60;40 &#956;m 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.

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...

Watch this Scientific Journal Video about Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays at JoVE.com

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

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.

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

The overall goal of this procedure is to simultaneously quantify multiple proteins on the chip using sandwich immunoassays in a spotter-free manner without cross reactivity between reagents. This method can help answer key questions in the quantitative proteomics field, such as the discovery and validation of candidate protein biomarkers in cancer. The main advantages of this technique are that it has cross reactivity between antibodies and antigen in multiplexed sandwich assays and that and users do not need a complex microarray spotter.Demonstrating the procedure will be Heidi Larkin, a scientific director at Parallex BioAssays. To begin preparing slides for the single-transfer method, fix a new transfer slide on the ink jet microarray spotter deck. Using a polystyrene microbead solution, or 30%glycerol, spot an array of alignment marks on the edge of the slide.Using the instrument software and spotter camera, take a picture of the alignment marks for quality control. Next fix a nitrocellulose or functionalized glass assay slide to the left of the transfer slide on an ink jet micro array spotter deck. Using alignment marks, spot 24 arrays of capture antibody solutions with 400 micrometer center-to-center spacing.Incubate the assay slide at room temperature in an atmosphere with 60%relative humidity for one hour. Then clip a 24-well silicone gasket onto the slide, aligned with the arrays. Pipette 80 microliters of a 0.1%solution of polysorbate 20 in PBS into each well.Shake the slide at 450 rpm for five minutes and discard the solution. Wash the slide in this way three times. Next load 80 microliters of BSA free-blocking solution into each rinsed well.Shake the slide at 450 rpm for one hour. Then discard the solution, remove the silicone gasket, and dry the assay slide using either nitrogen gas or a centrifuge. Seal the assay slide in an airtight bag with silica gel or another desiccant and store it at minus 20 degrees Celsius.Using the same alignment marks, spot 24 arrays of detection antibody solutions using 3.2 nanoliters per spot in a center-to-center spacing of 400 micrometers. Store the transfer slide at minus 20 degrees Celsius in a sealed bag with a desiccant. To begin preparing slides for the double-transfer method, fix a transfer slide on an ink jet microarray spotter deck.Spot 24 arrays of capture antibody solutions with the center-to-center spacing of 400 micrometers. Then turn the snap apparatus plunger switches 90 degrees to lock the plungers in the open position. Fit the capture transfer slide into the apparatus with the clipped corner against the fixed pogo pin.Release the pogo pins by turning the corresponding plunger switches back by 90 degrees. Ensure that the transfer slide is firmly held by the pogo pins and is flush with the fixed alignment pins. Then place a new nitrocellulose or a functionalized glass slide on the pogo pins with the coated side face down.Turn the other three switches to release the straight pins. Verify that both slides are held in place against the fixed alignment pins. Then fit the top shell onto the slide holder with the pillar seated in the alignment holes.Insert the closed apparatus into the snap cage and fully depress the closure tab. Leave the apparatus closed for one minute. Then pull up the closure tab to release the apparatus from the cage.Remove the top shell and separate the assay and transfer slides. Incubate the assay slide for one hour. Next clip a 24-well silicone gasket onto the slide and wash the slide three times.Then shake the slide with BSA-free blocking solution for one hour. Remove the gasket and dry the assay slide under a stream of nitrogen gas or a centrifuge. Next fix another transfer slide on the ink jet spotter deck.Spot 24 arrays of detection antibody solutions on the slide with 3.2 nanoliters per spot in a center-to-center spacing of 400 micrometers to make the transfer slide. Store the detection transfer slide at minus 20 degrees Celsius. To begin the immunoassay remove the prepared assay slide from the freezer and allow it to warm to room temperature in its sealed bag for 30 minutes.Prepare seven serially diluted solutions of proteins in PBS with 0.05%polysorbate 20. Prepare appropriately diluted sample solutions in the same buffer. Clamp a 24-well silicone gasket onto the room temperature assay slide.Load the seven protein dilutions into seven of the eight wells in a column. Load protein-free PBS with 0.05%polysorbate 20 into the last well of that column. Load the sample solutions into the remaining wells.Shake the slides at 450 rpm for one hour at room temperature or at 4 degrees Celsius overnight. Then wash the slide three times. Remove the gasket and dry the slide under a stream of nitrogen gas.Next allow the detection antibody transfer slide to warm to room temperature for 30 minutes in its sealed bag. Then incubate the slides for 20 minutes in a closed chamber with the relative humidity of 60%to rehydrate the arrays. Use the pogo pins to fix the detection transfer slide face up in the snap apparatus.Place the assay slide face down on the pogo pins and align the slide with the straight pins. Close the apparatus and snap transfer the detection antibodies to the assay slide. Remove the slides from the apparatus and incubate the assay slide for one hour.Clamp a 24-well silicone gasket to the slide and wash the slide three times. Then load into each well 80 microliters of a 2.5 microgram per milliliter solution of streptavidin fluoro-4 in PBS. Shake the slide at 450 rpm for 20 minutes.Wash the slide three times with a 0.1%solution of polysorbate 20 in PBS and once with distilled water. Then remove the gasket and dry the slide. Scan the slide with a fluorescent scanner and measure the spot intensities.Determine the limits of detection in the protein concentrations. Two fluorescent proteins were sequentially patterned on a nitrocellulose-coated slide using the double-transfer method with a 16-wells template. No cross-contamination was observed upon transferring the second fluorescent protein.Smaller spots with decreased spot-to-spot distance could be transferred to a glass slide with a highly hydrophobic surface. The double-transfer method resulted in minimal angular misalignment of the arrays. The double-transfer method was used to perform a 50-plex immunoassay of breast cancer-related proteins in the sandwich assay format.No cross reactivity was observed, allowing independent optimization of each antibody pair. Standard curves calculated from the assays indicated that 35 of the 50 proteins had picogram per milliliter limits of detection. Once mastered this technique can be completed in approximately three hours.This technique allows scientists to conduct microarray-based assay and to deliver reagents as nano droplets. While attempting this procedure, remember to fix the slides properly on the spotting deck and in the snap apparatus. After watching this video, you should have a good understanding of how to use the snap apparatus for cross-reactivity-free multiplexed sandwich immunoassays.

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Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...