The authors would like to acknowledge the startup fund from SUNY Albany and the access of facilities at the University at Albany Cancer Research Center.

Caution: Several chemicals used in this protocol are irritants and are hazardous in case of skin contact. Consult material safety data sheets (MSDS) and wear appropriate personal protective equipment before performing this protocol. The piranha solution used in Step (1.1.1) is highly corrosive and should be prepared by adding the peroxide slowly to the acid with agitation. Handle this solution with extreme caution in a fume hood. Use appropriate eye protection and acid-resistant gloves. Trimethylchlorosilane (TMCS) is a ...

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+technologies+for+monitoring+immune+systems.">Single-cell technologies for monitoring immune systems.</a> Nat Immunol. 15 (2), 128-135 (2014).</li><li>Varadarajan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+efficient+functional+characterization+and+recovery+of+HIV-specific+human+CD8(+)+T+cells+using+microengraving.">Rapid, efficient functional characterization and recovery of HIV-specific human CD8(+) T cells using microengraving.</a> Proc Natl Acad Sci USA. 109 (10), 3885-3890 (2012).</li><li>Ma, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation-based+delivery+of+cell-penetrating+peptide+conjugates+of+peptide+nucleic+acids+for+antisense+inhibition+of+intracellular+bacteria.">Electroporation-based delivery of cell-penetrating peptide conjugates of peptide nucleic acids for antisense inhibition of intracellular bacteria.</a> Integr Biol-Uk. 6 (10), 973-978 (2014).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitating+cell-cell+interaction+functions+with+applications+to+glioblastoma+multiforme+cancer+cells.">Quantitating cell-cell interaction functions with applications to glioblastoma multiforme cancer cells.</a> Nano Lett. 12 (12), 6101-6106 (2012).</li><li>Kravchenko-Balasha, N., Wang, J., Remacle, F., Levine, R. 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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+proteomic+chip+for+profiling+intracellular+signaling+pathways+in+single+tumor+cells.">Single-cell proteomic chip for profiling intracellular signaling pathways in single tumor cells.</a> Proc Natl Acad Sci USA. 109 (2), 419-424 (2012).</li><li>Ma, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clinical+microchip+for+evaluation+of+single+immune+cells+reveals+high+functional+heterogeneity+in+phenotypically+similar+T+cells.">A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells.</a> Nat Med. 17 (6), 738-743 (2011).</li><li>Deng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+microfluidic+chip+system+for+single-cell+secretion+profiling+of+rare+circulating+tumor+cells.">An integrated microfluidic chip system for single-cell secretion profiling of rare circulating tumor cells.</a> Sci Rep. 4, 7499 (2014).</li><li>Vermesh, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-density,+multiplexed+patterning+of+cells+at+single-cell+resolution+for+tissue+engineering+and+other+applications.">High-density, multiplexed patterning of cells at single-cell resolution for tissue engineering and other applications.</a> Angew Chem Int Ed. 50 (32), 7378-7380 (2011).</li><li>Yu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics-based+single-cell+functional+proteomics+for+fundamental+and+applied+biomedical+applications.">Microfluidics-based single-cell functional proteomics for fundamental and applied biomedical applications.</a> Annu Rev Anal Chem. 7, 275-295 (2014).</li><li>Bailey, R. C., Kwong, G. A., Radu, C. G., Witte, O. N., Heath, J. 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Flow pattern design is the first critical step in fabricating the 2-D microarray. To generate two overlapping DNA patterns on a glass substrate, the channel features of the first design should be perpendicular to those of the second (Figure 1A-B). The designs also consider the downstream applications of the microarray. In the case of single cell analysis, the microarray is used to detect proteins from single cells enclosed in microchambers, therefore the channel dimensions are made compatible wi...

Antibody microarrays have been widely used in proteomic studies for decades to examine the presence of targeted proteins, including protein biomarkers1-3. Although this field is currently facing great challenges from other high-throughput technologies such as mass spectrometry (MS), there is still plenty of room for the utility of antibody microarrays, mainly because these devices afford simple data interpretation and easy interface with other assays. In recent years, the integration of microarrays into microchip scaffolds has provided the antibody microarray a new opportunity to thrive4-7. For instance, the barcode microarray integrated into a single-cell microchip has been used in cell communication studies8,9. This technology has distinctive advantages over other available microarray technologies. It features array elements at 10-100 &#956;m, much smaller than the typical 150 &#956;m size used in conventional microarray elements. The construction of smaller array elements is achieved using systematic flow-patterning approaches, and this gives rise to compact microarrays that can detect single-cell secreted proteins and intracellular proteins. Another advantage is the use of a simple, instrument-free setup. This is particularly important, because most laboratories and small companies may not be able to access microarray core facilities. Such barcode antibody microarrays feature enhanced assay throughput and can be used to perform highly multiplexed assays on single cells while achieving high sensitivity and specificity comparable with that of conventional sandwich enzyme-linked immunosorbent assay (ELISA8). This technology has found numerous applications in detecting proteins from glioblastoma9-11, T cells12, and circulating tumor cells13. Alternatively, barcode DNA microarrays alone have been utilized in the precise positioning of neurons and astrocytes for mimicking the in vivo assembly of brain tissue14.
This protocol focuses only on the experimental steps and build-up blocks of the two-dimensional (2-D) barcode antibody microarray which has potential applications in the detection of biomarkers in fluidic samples and in single cells. The technology is based on an addressable single-stranded, one-dimensional (1-D) DNA microarray constructed using orthogonal oligonucleotides that are patterned spatially on glass substrates. The 1-D pattern is formed when parallel flow channels are used in the flow-patterning step, and such a pattern appears as discrete bands visually similar to 1-D Universal Product Code (UPC) barcodes. The construction of a 2-D (n x m) antibody array &#8212; reminiscent of a 2-D Quick Response (QR) matrix code&#160;&#8212; needs more complex patterning strategies, but allows for the immobilization of antibodies at a higher density8,15. The fabrication requires two DNA patterning steps, with the first pattern perpendicular to the second. The points of intersection of these two patterns constitute the n x m elements of the array. By strategically selecting the sequences of single-stranded DNA (ssDNA) utilized in flow-patterning, each element in a given array is assigned a specific address. This spatial reference is necessary in distinguishing between fluorescence signals on the microarray slide. The ssDNA array is converted into an antibody array through the incorporation of complementary DNA-antibody conjugates, forming a platform called DNA-encoded antibody library (DEAL16).
This video protocol describes the key steps in creating n x m antibody arrays which include preparing polydimethylsiloxane (PDMS) barcode molds, flow-patterning ssDNA in two orientations, preparing antibody-oligonucleotide DEAL conjugates, and converting the 3 x 3 DNA array into a 3 x 3 antibody array.

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			<td height="21" style="height:21px;width:263px;">Sylgard 184 silicone elastomer base</td>
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			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:263px;">Sylgard 184 silicone elastomer curing agent</td>
			<td style="width:403px;">Dow Corning</td>
			<td style="width:119px;">3097358-1004</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">SU-8 2025 photoresist</td>
			<td style="width:403px;">MicroChem</td>
			<td style="width:119px;">Y111069</td>
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			<td height="21" style="height:21px;width:263px;">Silicon wafers&#160;</td>
			<td style="width:403px;">University Wafers</td>
			<td style="width:119px;">452</td>
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			<td height="21" style="height:21px;width:263px;">Poly-L-lysine coated glass slides</td>
			<td style="width:403px;">Thermo Scientific</td>
			<td style="width:119px;">C40-5257-M20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Oligonucleotides</td>
			<td style="width:403px;">Integrated DNA Technologies</td>
			<td style="width:119px;"></td>
			<td>*Custom-ordered from Integrated DNA Technologies, see table below</td>
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			<td height="21" style="height:21px;width:263px;">Poly-L-lysine adhesive stock solution</td>
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			<td style="width:119px;">1339</td>
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			<td height="21" style="height:21px;width:263px;">Bis (sulfosuccinimidyl) suberate&#160; (BS3)</td>
			<td style="width:403px;">Thermo Scientific</td>
			<td style="width:119px;">21585</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">1x Phosphate buffered saline, pH 7.4</td>
			<td style="width:403px;">Quality Biological</td>
			<td style="width:119px;">114-058-101</td>
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			<td height="40" style="height:40px;width:263px;">&#196;kta Explorer 100 Fast Protein Liquid Chromatography (FPLC) System</td>
			<td style="width:403px;">GE (Amersham Pharmacia)&#160;</td>
			<td>18-1112-41</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Superose 6 10/300 GL column</td>
			<td style="width:403px;">GE Healthcare Life Sciences</td>
			<td>17-5172-01</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Capture antibodies</td>
			<td style="width:403px;">various</td>
			<td style="width:119px;">various</td>
			<td>*Antibody selection depends on application</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:263px;">Succinimidyl-6-hydrazino-nicotinamide (S-HyNic)</td>
			<td style="width:403px;">Solulink</td>
			<td style="width:119px;">S-1002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Succinimidyl-4-formylbenzamide (S-4FB)</td>
			<td style="width:403px;">Solulink</td>
			<td style="width:119px;">S-1004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">N,N-dimethylformamide</td>
			<td style="width:403px;">Sigma-Aldrich</td>
			<td style="width:119px;">227056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Citric acid, anhydrous</td>
			<td style="width:403px;">Acros</td>
			<td style="width:119px;">42356</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Sodium hydroxide</td>
			<td style="width:403px;">Fisher Scientific</td>
			<td style="width:119px;">S318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Amicon Ultra spin filter 10 kDa MWCO</td>
			<td style="width:403px;">EMD Millipore</td>
			<td>UFC201024</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Spin coater</td>
			<td style="width:403px;">Laurell Technologies</td>
			<td>WS-650-MZ</td>
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			<td height="21" style="height:21px;">Biopsy punch with plunger (0.50 mm diameter)</td>
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			<td align="right" style="width:119px;">57393</td>
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			<td height="21" style="height:21px;">Trimethylchlorosilane</td>
			<td style="width:403px;">Sigma Aldrich</td>
			<td align="right" style="width:119px;">92361</td>
			<td></td>
		</tr>
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flow-pattern guided fabrication of high-density barcode antibody microarray

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications.

The designs for the PDMS molds (Figure 1A-1B) were drawn using a CAD program (AutoCAD). Two designs shown feature channels for flow patterning, one horizontal and one vertical. The left and right parts of each design are symmetric; either of them could be inlets or outlets. Each of 20 channels is winding from one end all the way to the other end. Each design is printed on a chrome photomask (Figure 1C). The fabricated SU-8 master on a wafer is shown in <s...

Watch this Scientific Journal Video about Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray at JoVE.com

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

This protocol outlines the fabrication of a large-scale, multiplexed two-dimensional DNA or antibody array, with potential applications in cell signaling studies and biomarker detection.

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this ...

The overall goal of this methodology is to fabricate an antibody array with applications in single-cell analysis. This method can help answer key questions in the field of single-cell analysis such as how can we detect secret proteins and intracellular proteins simultaneously from the same single cell. And how can we fabricate a micro-array platform to accomplish that objective.The main advantage of this technique is that it is customizable. By varying the flow patterning strategies, arrays of various dimensions may be created. In advance, generate the SU-8 master for the barcode flow patterns.Instructions are provided in the text protocol. This involves designing, and making a chrome photo-mask. Placing the mask on a photo-resist coat and applying UV provides the pattern.To start preparing the PDMS barcode mold, combine 40 grams of silicon elastomer base with four grams of curing agent, then stir vigorously for 10 minutes. Then, de-gas the pre-polymer solution for 20 minutes under vacuum. Next, load the solution into a petri dish containing a silicon wafer with the SU-8 master of the barcode pattern.Pour the solution to a depth of 7.45 millimeters. Then, de-gas the mixture in the dish to remove any remaining bubbles. Follow the de-gassing by baking the mixture for an hour at 75 Degrees Celsius to cure the PDMS.Next, cutting with a scalpel, carefully remove the barcode mold. Then, carefully peel the slab from the wafer. Next, trim the edges of the slab to make the desired shape for the mold.Finish the mold by using a biopsy punch with plunger to punch out half millimeter holes at the circular features on the pattern which represent inlets and outlets. After blowing any dust off a poly-L-lysine coated glass slide with a nitrogen blowgun, attach the PDMS mold to the slide. Carefully line up the edges of the mold to the slide.Now, bake the assembly for 90 minutes at 75 degrees Celsius to strengthen the bond between the PDMS and the slide. Meanwhile, prepare 20 pieces of flexible polyethylene tubing for the inlets and outlets. On each piece of tubing, attach a stainless steel hollow pin with a one millimeter diameter.Then, via the pins, fill the lengths with a centimeter or more of poly-L-lysine. Once bonded to the glass, carefully fasten the pins to the inlets of the mold. This is the most difficult step of the preparation.Do not rush the process. Attach the other end of the tubes to a pressure-regulated nitrogen tank setup which consists of a network of 20 three-way valves and can handle two molds at once. Using the setup, blow solution through the mold at 0.5 to one PSI for at least six hours.Begin by aspirating freshly prepared single-stranded DNA solutions in BS-3 through the stainless steel pins and into the polyethylene tubing. Then, couple the PDMS mold to the nitrogen source and flow the solution through the barcode mold for about 40 minutes or until all the channels are filled. Then, stop the flow and let the mold incubate at room-temperature for two hours.After a couple of hours, bake the barcode mold at 75 degrees Celsius for an hour. Once baked, detach the mold from the slide and gently wash the slide with 01%SDS. Then, wash the slide three times with ultra-pure water.Follow the washes by drying the barcode slide using a slide-spinner. Once dry, store the barcode slide in a clean 50 milliliter tube. To proceed, follow the text protocol to validate the one-dimensional pattern.To perform the validation procedure, block the slide, hybridize cyanine three complementary DNA to it and measure the results. The SU-8 master for a three by three array has a flow pattern perpendicular to that of the first design. Use the previously described method to make a new PDMS mold of this pattern.To start, make fresh working concentrations of the nine oligonucleotides. Now, flow a three percent BSA blocking solution into all 20 channels of the array slide for an hour at 0.5 to one PSI. Next, load the DNA solutions into their respective channels.After flowing them for about 40 minutes, incubate the slide at room-temperature for two hours to allow the DNA to hybridize. Next, flow three percent BSA blocking solution into all 20 channels for an hour. This will remove the non-hybridized DNA.Now, peel the array slide from the PDMS and dip the slide into three percent BSA once. Then, dip the slide into PBS twice followed by one dip in two percent PBS. Then, as before, dry the slide using a microscope slide spinner.To proceed, perform a validation step similar to validating one-dimensional slides. Use oligonucleotides one-prime to nine-prime all conjugated to cyanine three. Save the three by three array slide in a 50 milliliter centrifuge tube for subsequent use.The text protocol further explains how to convert the DNA array into an antibody array. After preparing and purifying the antibody-oligo conjugates, prepare another PDMS mold with micro-chambers. See the text for details.Mate this mold with the array slide. Now, block the slide with one percent BSA and incubate it for an hour at room temperature. During the incubation, prepare 200 micro-liters of antibody-oligonucleotide conjugates.After the block, add the antibody-oligo cocktail to the setup. Let the antibodies incubate on the array for an hour at 37 degrees Celsius, and then clean and dry the slide before proceeding with slide dips described in the text protocol. After following the described protocol, using a three by three array, the resulting antibody panel was used in multi-plex detection, mainly through sandwich ELISA platform.The proteins showed less than 10%variation from one end to the other end of the glass slide. On the three by three array, up to seven proteins can be detected along with a reference labeled by cyanine three and a negative control. In a single-cell experiment performed for a tumor study, the six proteins were all detected simultaneously.For calibration, recombinant proteins were applied to an array at various concentrations. The sensitivity of detection was quite similar to a conventional sandwich ELISA. While attempting this procedure, its important to remember to keep the PDMS molds free of particulate matter.Dust in the channels can result in arrays with poor uniformity and low sensitivity. After its development, this technique paved the way for researchers in the field of cancer research to explore cell-cell signaling using models of tumor micro-environments.

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8.8K 次观看.  University at Albany, State University of New York. The overall goal of this methodology is to fabricate an antibody array with applications in single-cell analysis. This method can help answer key questions in the field of single-cell analysis such as how can we detect secret proteins and intracellular proteins simultaneously from the same single cell. And how can we fabricate a micro-array platform to accomplish that objective.The main advantage of this technique is that it is customizable. By varying the flow patterning strategies, a...