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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Cell. proteomics. 11 (4), (2012).</li><li>Li, H., Bergeron, S., Juncker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray-to-Microarray+Transfer+of+Reagents+by+Snapping+of+Two+Chips+for+Cross-Reactivity-Free+Multiplex+Immunoassays.">Microarray-to-Microarray Transfer of Reagents by Snapping of Two Chips for Cross-Reactivity-Free Multiplex Immunoassays.</a> Anal. Chem. 84 (11), 4776-4783 (2012).</li><li>Li, H., Munzar, J. D., Ng, A., Juncker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+snap+chip+for+high-density+sub-nanoliter+chip-to-chip+reagent+transfer.">A versatile snap chip for high-density sub-nanoliter chip-to-chip reagent transfer.</a> Sci. Rep. 5, 11688 (2015).</li><li>Li, H., Bergeron, S., Annis, M. G., Siegel, P. M., Juncker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+analysis+of+38+proteins+during+the+progression+of+human+breast+tumor+in+mice+using+an+antibody+colocalization+microarray.">Serial analysis of 38 proteins during the progression of human breast tumor in mice using an antibody colocalization microarray.</a> Mol. Cell. Proteomics. 14, 1024-1037 (2015).</li><li>Bergeron, S., Laforte, V., Lo, P. S., Li, H., Juncker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+mixtures+of+14+hygroscopic+additives+to+improve+antibody+microarray+performance.">Evaluating mixtures of 14 hygroscopic additives to improve antibody microarray performance.</a> Anal. Bioanal. Chem. 407 (28), 8451-8462 (2015).</li><li>Whiteaker, J. 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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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Bioengineering

6.4K ビュー.  McGill University and Génome Québec Innovation Centre. 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 ...

ビデオ: 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...

a snap chip technology for a cross-reactivity-free multiplexed sandwich immunoassay

The video showcases the implementation of snap chip technology for cross-reactivity-free multiplexed sandwich immunoassays. The simple action of snapping two microarray slides, each containing different reagents, ensures reagent transfer without cross-contamination and helps to detect multiple proteins using sandwich...

A Snap Chip Technology for a Cross-Reactivity-Free Multiplexed Sandwich Immunoassay

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...

production of nurr-1 specific polyclonal antibodies free of cross-reactivity against its close homologs, nor1 and nur77

Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs, Nor1 and Nur77

Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by...

multimodal cross-device and marker-free co-registration of preclinical imaging modalities

Author Spotlight: An Efficient and Robust Software for Automated Fusion of Multiple Preclinical Imaging Modalities

The combination of multiple imaging modalities is often necessary to gain a comprehensive understanding of pathophysiology. This approach utilizes phantoms to generate a differential transformation between the coordinate systems of two modalities, which is then applied for co-registration. This method eliminates the need for fiducials in production...

Multimodal Cross-Device and Marker-Free Co-Registration of Preclinical Imaging Modalities

pip-on-a-chip: a label-free study of protein-phosphoinositide interactions

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH...

multimodal analytical platform on a multiplexed surface plasmon resonance imaging chip for the analysis of extracellular vesicle subsets

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

This paper proposes a new generation of multiparametric analytical platforms with increased throughput for the characterization of extracellular vesicle subsets. The method is based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy, coupled with Raman spectroscopy, to qualify vesicular targets trapped on a microarray biochip.

collection of serum- and feeder-free mouse embryonic stem cell-conditioned medium for a cell-free approach

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

This protocol provides a method for the collection of mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) derived from serum (fetal bovine serum, FBS)- and feeder (mouse embryonic fibroblasts, MEFs)-free conditions for a cell-free approach. It may be applicable for the treatment of aging and aging-associated...

a model of free tissue transfer: the rat epigastric free flap

A Model of Free Tissue Transfer: The Rat Epigastric Free Flap

This paper describes the steps required to raise a fasciocutaneous epigastric free flap and transfer it to the neck in the...

defined xeno-free and feeder-free culture conditions for the generation of human ipsc-derived retinal cell models

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical...

fabrication of three-dimensional paper-based microfluidic devices for immunoassays

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

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based...

protocol for her2 fish using a non-cross-linking, formalin-free tissue fixative to combine advantages of cryo-preservation and formalin fixation

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

Fluorescence in-situ hybridization (FISH) is often required in combination with histopathology and molecular diagnostics for selection of therapy in personalized medicine. A novel non-cross-linking, formalin-free tissue fixative that allows high quality morphologic, molecular and FISH analyses from the same specimen by addition of a post-fixation step before FISH is...

a microfluidic-based electrochemical biochip for label-free dna hybridization analysis

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8...

procedure for the development of multi-depth circular cross-sectional endothelialized microchannels-on-a-chip

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular...

free-floating immunostaining of mouse brains

Free-floating Immunostaining of Mouse Brains

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and...

sealable femtoliter chamber arrays for cell-free biology

Sealable Femtoliter Chamber Arrays for Cell-free Biology

A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene...

antibody-free assay for rna methyltransferase activity analysis

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is...

from cells to cell-free protein synthesis within 24 hours using cell-free autoinduction workflow

From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow

This work describes the preparation of cell extract from Escherichia coli (E. coli) followed by cell-free protein synthesis (CFPS) reactions in under 24 hours. Explanation of the cell-free autoinduction (CFAI) protocol details improvements made to reduce researcher oversight and increase quantities of cell extract obtained.

microfluidic buffer exchange for interference-free micro/nanoparticle cell engineering

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound...

cell-free protein synthesis system for building synthetic cells

Author Spotlight: Optimizing CFPS Systems for Synthetic Cell Construction

This protocol describes the Cell-Free Protein Synthesis (CFPS) system used in constructing synthetic cells. It outlines key stages with representative results in different micro-compartments. The protocol aims to establish best practices for diverse laboratories in the synthetic cell community, advancing progress in synthetic cell...

Cell-Free Protein Synthesis System for Building Synthetic Cells

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration1,2 and efficient separation and extraction based on ionic mobility.3 For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation4-8 and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood.9-12
ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.13). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species.
We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation.
We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.

Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

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 bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

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 mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

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. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

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

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

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 spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

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 research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

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 well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

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