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 corrosive, flammable chemical used in an optional step after (1.1.6). Handle this chemical in a fume hood.
Note: Perform the barcode slide fabrication and critical flow-patterning procedures in a clean room to minimize contamination by particulate matter. Dust particles may block the ports and microchannels of PDMS molds and interfere with flow-patterning.
1. Construction of the One-dimensional DNA Barcode Slide
<ol>
	<li>Preparation of the SU-8 master for barcode flow patterns 
	Note: The drawings for the perpendicular flow patterns (Figure 1A-B) are created using a computer-aided design (CAD) software. These patterns are rendered on a chrome photomask. Transparent areas of the mask correspond to the features of the SU-8 master.
	<ol>
		<li>Clean a silicon wafer (100 mm diameter) thoroughly in a mixture of 3 H2SO4: 1 30% H2O2 (piranha solution) heated to 96&#160;&#176;C. Wash the wafer with deionized water and isopropyl alcohol, followed by drying with a nitrogen blow gun.</li>
		<li>Pour about 4 ml of SU-8 2025 photoresist on the wafer. Use a programmable spin coater to uniformly spread the photoresist on the wafer for 10 sec at 500 rpm, then 30 sec at 3,000 rpm. This creates a photoresist layer with a thickness of ~25 &#181;m. Gradually allow spinning to slow down before stopping &#8212; this is to maintain an even coating on the wafer&#39;s surface.</li>
		<li>Bake the coated wafer on a hotplate for 1 min at 65 &#176;C, then for 5 min at 95 &#176;C. This step allows the coating to solidify. Cool to RT for 5 min.</li>
		<li>Place the chrome mask (Figure 1C) on the photoresist coat. Expose the mask features to near-UV light (350-400 nm, exposure energy 150-160 mJ/cm2) for 20 sec. 
		Note: The design on the chrome mask contains 20 channels, each of which are 20 &#181;m wide with 50 &#181;m pitch. The channels are winding from one end of the pattern to the opposite end. Altogether, the 20 channels cover a rectangular area with a length of ~40 mm and a width of ~20 mm. Each channel is flanked by two circular features which correspond to an inlet and an outlet. Inlets and outlets are interchangeable.</li>
		<li>Bake the exposed wafer on a hotplate for 5 min at 95 &#176;C. Cool to RT gradually.</li>
		<li>Immerse the wafer in SU-8 developer with agitation for 5 min. Wash the wafer with a small portion of fresh SU-8 developer, followed by isopropyl alcohol. Dry the wafer using a nitrogen blow gun. Hard-bake the wafer on a 200 &#176;C hotplate for 30 min, and allow the wafer to cool gradually to RT. 
		Note: Development may be carried out for a longer time if a white film is observed after washing in this step. 
		Optional: Silanize the SU-8 master by exposing it to trimethylchlorosilane vapor in a closed Petri dish for 10 min.</li>
	</ol>
	</li>
	<li>Preparation of the PDMS barcode mold
	<ol>
		<li>Combine 40.0 g silicone elastomer base with 4.0 g curing agent. Stir the prepolymer mixture vigorously for 10 min. Degas for 20 min under vacuum. 
		Note: As a general rule, use a 10:1 (base: curing agent) mass ratio.</li>
		<li>Pour the prepolymer into a Petri dish containing a silicon wafer with the SU-8 master of the barcode pattern. The height of the PDMS mixture should be about 7.5 mm or above. Degas the mixture in the Petri dish for a second time to remove any remaining bubbles, then bake the mixture for 1 hr at 75 &#176;C to allow PDMS to cure. 
		Note: It is important to maintain enough thickness of the PDMS slab at ~7.5 mm or above to avoid adding too much tension to the PDMS-glass bond upon insertion of pins/tubing through holes in the PDMS mold in step (1.3.3.1)</li>
		<li>Using a scalpel, carefully cut around the area of the PDMS slab that contains the barcode mold features and peel the slab from the wafer.</li>
		<li>Trim the edges of the slab to attain the desired shape of the PDMS barcode mold. Punch 20 holes (1.0-mm diameter) through the mold using a biopsy punch with plunger. Ensure that the holes are aligned with the circular features of the barcode pattern. These holes serve as the inlets and outlets.</li>
	</ol>
	</li>
	<li>One-dimensional patterning of poly-L-lysine (PLL)
	<ol>
		<li>Remove any dust on the surface of a poly-L-lysine coated glass slide using a nitrogen blow gun. Attach the PDMS mold to the clean glass slide. Ensure that the edges of the mold and the slide are aligned.</li>
		<li>Bake for 1.5 hr at 75 &#176;C to strengthen the bond between the PDMS mold and the PLL-coated slide. Meanwhile, prepare 20 pieces of flexible polyethylene tubing (3- to 4-inch pieces, with an inner diameter of 0.5 mm and an outer diameter of 1.5 mm). 
		Note: The number of pieces of tubing corresponds to the number of inlets in the PDMS barcode mold. The tubing serves to couple the channels on the PDMS barcode mold to a nitrogen gas tank equipped with a pressure regulator.</li>
		<li>To one end of each piece of tubing, attach a stainless steel hollow pin (1-mm diameter). Aspirate sterile-filtered poly-L-lysine solution through the pin, until at least 1 cm of the tubing is filled with the solution.
		<ol>
			<li>Fasten the pins (connected to solution-filled tubing) to the inlets of the PDMS barcode mold (Figure 1E). Attach the other end of the tubes to a pressure-regulated nitrogen tank set-up. Allow the solution to flow through the mold using a pressure range of 0.5-1 psi for at least 6 hr. 
			Note: Refer to step (1.3.3) for all flow-patterning procedures.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>One-dimensional patterning of ssDNA 14 
	Note: The phosphate buffered saline (PBS) used in subsequent steps is prepared from 137 mM NaCl, 10 mM Na2HPO4, and 2 mM KH2PO4. The pH of the buffer is 7.4.
	<ol>
		<li>Prepare 2 mM bis(sulfosuccinimidyl)suberate (BS3) solution in PBS. Prepare 300 &#181;M solutions of A, B, and C ssDNA (5&#39;-amine modified, 80 nucleotides) in PBS or ultrapure water. 
		Note: Use the BS3 solution within about 30 min after preparation because the N-hydroxysuccinimide ester easily undergoes hydrolysis.</li>
		<li>Combine 2.5 &#181;l of 300 &#181;M A, B, or C ssDNA with 2.5 &#181;l of 2 mM bis(sulfosuccinimidyl)suberate in PBS for each channel. A, B, and C DNA are designated to channels 1, 2, and 3, respectively. This order is also applied to remaining sets of 3 channels (Figure 2A).</li>
		<li>Perform step (1.3.3). This time, aspirate the 5-&#181;l BS3/DNA solutions through stainless steel pins and into the polyethylene tubing, then couple the PDMS mold to the nitrogen source. Allow the BS3/DNA solution to flow through the barcode mold for about 40 min&#160;or until at the channels are filled.</li>
		<li>Stop the flow once all channels are filled, then incubate the BS3/DNA solution in the barcode at RT for 2 hr. Do not allow the solution to dry up. Bake the PDMS mold with attached barcode slide for 1 hr at 75 &#176;C. 
		Note: To facilitate alignment in subsequent flow-patterning steps, the edges of the channel pattern on the barcode slide may be outlined. This is done by carefully scratching the glass surface using a diamond scribe to generate alignment markers on the bottom of the glass slide. Alignment in later stages of the protocol may be checked under a microscope.</li>
		<li>Remove the PDMS mold from the barcode slide. Wash the slide gently with 0.01% SDS once and three times with ultrapure water.
		<ol>
			<li>Dry the barcode slide using a microscope slide spinner. Store the barcode slide in a clean 50-ml centrifuge tube for subsequent use.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Validation of the One-dimensional Pattern on the Barcode Slide
Note: This validation protocol may also be adapted for use in assessing the quality of subsequent flow patterning steps.
<ol>
	<li>Blocking of the slide
	<ol>
		<li>Prepare 1% bovine serum albumin (BSA) in PBS. Filter this solution through a 0.45 &#181;m syringe filter before use. Using a gel-loading tip, apply 50 &#181;l of 1% BSA solution to one edge of the barcode slide.</li>
		<li>Incubate the 1% BSA solution for 1 hr at RT.</li>
	</ol>
	</li>
	<li>Incubation with Cyanine 3 (Cy3)-conjugated complementary DNA
	<ol>
		<li>Prepare a cocktail of A&#39;, B&#39;, and C&#39; oligonucleotides conjugated to Cy3 at one end. The sequences of A&#39;, B&#39;, and C&#39; are complementary to those of A, B, and C, respectively. The working concentration of the Cy3-DNA is 0.05 &#181;M in 0.1% BSA.</li>
		<li>Remove the BSA solution from the slide by pipetting, then apply 30 &#181;l of the DNA cocktail solution on the same edge of the slide. Incubate the Cy3-DNA cocktail on the slide at RT for 1 hr. Perform this incubation step in the dark to protect the Cy3 moiety from photobleaching.</li>
	</ol>
	</li>
	<li>Analysis of fluorescence intensity
	<ol>
		<li>Pipette out the Cy3-DNA cocktail and wash the slide in 1% BSA, PBS, and finally in diluted PBS (1 part PBS with 50 parts ultrapure water). Dry the slide using a spinner.</li>
		<li>Observe the fluorescence (Figure 2B) using a fluorescence microscope or a microarray scanner. When using a microarray scanner, set the laser emission wavelength to 532 nm, the pixel size at 5 microns, photomultiplier tube (PMT) gain at 450 and power at 15%.</li>
	</ol>
	</li>
</ol>
3. Fabrication of the 2-dimensional (3x3) DNA Array14
<ol>
	<li>Preparation of the PDMS barcode mold
	<ol>
		<li>Perform procedure (1.1) to construct another SU-8 master, but this time use a chrome mask with a flow pattern perpendicular to that of the first design. Perform procedure (1.2) to construct a new PDMS mold with the perpendicular pattern.</li>
	</ol>
	</li>
	<li>Two-dimensional flow patterning of ssDNA
	<ol>
		<li>For a 3 x 3 array, prepare 150 &#181;M stock solutions of A&#39;-i, B&#39;-ii, C&#39;-iii, A&#39;-iv, B&#39;-v, C&#39;-vi, A&#39;-vii, B&#39;-viii, and C&#39;-ix DNA in 3% BSA/PBS. These oligonucleotides serve as &#34;bridging sequences&#34; (Figure 2A). Combine the oligonucleotide solutions (Solutions 1 to 3) such that the working concentration is 50 &#181;M for each oligonucleotide. Use the guide provided in Table 1.</li>
		<li>Flow 3% BSA/PBS blocking solution into all 20 channels for 1 hr at 0.5-1 psi.</li>
		<li>Flow Solutions 1, 2, and 3 into channels 1, 2, and 3, respectively. Follow this order for subsequent sets of 3 channels. Flow is usually completed in around 40 min. Incubate the DNA solutions at RT for 2 hr to allow the DNA to hybridize.</li>
		<li>Flow 3% BSA/PBS blocking solution into all 20 channels for 1 hr at 0.5-1 psi to remove unhybridized DNA. Peel off the PDMS slab, and wash the resulting DNA microarray slide by dipping the glass slide into 3% BSA/PBS once and PBS twice, followed by diluted PBS (1 part PBS and 50 parts ultrapure water). Dry the slide using a microscope slide spinner.</li>
		<li>Perform a second validation step (Figure 2C) similar to that in section 2. Use oligonucleotides i&#39; to ix&#39; that are Cy3-conjugated. Store the 3 x 3 array slide in a 50-ml centrifuge tube for subsequent use. 
		Note: The DNA microarray can be stored at RT in a desiccator for months.</li>
	</ol>
	</li>
</ol>
4. Conversion of the 3 x 3 DNA Array into an Antibody Array
<ol>
	<li>Preparation of antibody-oligonucleotide (DEAL) conjugates
	<ol>
		<li>Prepare solutions of 200 mM succinimidyl-4-formylbenzoate (S-4FB) and 40 mM succinimidyl-6-hydrazinonicotinamide (S-HyNic) in anhydrous N,N-dimethylformamide (DMF).</li>
		<li>Prepare up to 7 separate solutions of capture antibodies (1 mg/ml) in PBS. 
		Note: If the antibody stock solutions contain sodium azide bacteriostat, perform buffer exchange with PBS using spin desalting columns with 7 kDa molecular weight cut-off (MWCO).</li>
		<li>Prepare separate 400 &#181;M solutions of i&#39;, ii&#39;, iii&#39;, iv&#39;, v&#39;, vi&#39;, and vii&#39; DNA. Assign each capture antibody to one oligonucleotide sequence. Combine 40 &#181;l of 400 &#181;M DNA with 12.25 &#181;l of DMF in microcentrifuge tubes and spin down to mix thoroughly.
		<ol>
			<li>To each DNA/DMF solution, add 2.3 &#181;l of 200 mM S-4FB in DMF. For every 100 &#181;g of capture antibody, add 2.25 &#181;l of 40 mM S-HyNic in DMF.</li>
		</ol>
		</li>
		<li>Incubate the antibody/S-HyNic and DNA/S-4FB solutions for 4 hr at RT.</li>
		<li>In preparation for the antibody-DNA coupling reaction, prepare new spin desalting columns (one for each DNA solution and one for each antibody) by washing them with citrate buffer at pH 6.</li>
		<li>After 4 hr of incubation, perform buffer exchange for each antibody/S-HyNic and DNA/S-4FB solution. Combine the S-4FB-conjugated i&#39;, ii&#39;, iii&#39;, iv&#39;, v&#39;, vi&#39; and vii&#39; oligonucleotides with the antibody-S-HyNic conjugates. Incubate the mixtures for 2 hr at RT.</li>
		<li>Incubate the reaction O/N at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Purification of antibody-oligonucleotide (DEAL) conjugates 
	Note: To purify the antibody-oligonucleotide conjugates, perform fast protein liquid chromatography (FPLC) on a standard FPLC system equipped with a Superose 6 10/300 GL column.
	<ol>
		<li>Set the wavelength of UV detector of the FPLC system to 280 nm. Use isocratic flow of PBS (pH 7.4) at 0.3 ml/min to separate the antibody-oligonucleotide conjugates from the excess S-4FB-DNA.</li>
		<li>Pool the fractions containing the conjugate and concentrate the fractions to a volume of 150 &#181;l using spin filters with a 10 kDa MWCO.</li>
	</ol>
	</li>
	<li>Two-dimensional patterning of antibodies
	<ol>
		<li>Prepare another PDMS mold with microchambers or micro-wells using fabrication procedures in step (1.2). The PDMS mold may contain microliter to nanoliter wells for immunoassays. 
		Note: For general biomarker detection in fluidic samples, a PDMS mold with multiple wells is mated with the array slide. The features of the PDMS mold depend on the system being studied. For instance, the detection of proteins from single cell experiments can be performed with a PDMS mold containing microchambers with 0.15-nl&#160;volumes.</li>
		<li>(Optional) Subject the PDMS mold to plasma cleaning (18 W) for 1.5 min to render its patterned surface hydrophilic. Prior to plasma cleaning, use adhesive tape to block all other surfaces that will be directly bonded to the 3 x 3 array slide. 
		Note: Step (4.3.2) is optional but is usually performed when the PDMS mold is intended for use in single cell experiments.</li>
		<li>Mate the PDMS mold with the 3 x 3 array slide, then block the slide with 1% BSA in PBS. Incubate for 1 hr at RT. Meanwhile, prepare a cocktail (200 &#181;l final volume) of antibody-oligonucleotide conjugates. The working concentration of each conjugate is 10 &#181;g/ml in 1% BSA/PBS.</li>
		<li>Add the antibody-oligonucleotide cocktail, then incubate for 1 hr at 37 &#176;C to allow the oligonucleotide moiety of the conjugates to hybridize with specific spots on the 3 x 3 arrays, thereby converting the DNA array to an antibody array.</li>
		<li>Repeat step (3.2.4) to clean and dry the slide. The highest sensitivity can be achieved if the antibody array is used immediately. Prolonged storage may result in loss of sensitivity.</li>
	</ol>
	</li>
	<li>Detection of proteins
	<ol>
		<li>Reconstitute recombinant proteins or prepare a filtered cell sample.</li>
		<li>Mate the microarray with a custom-designed chip, and block the surface with 3% BSA in PBS for 1 hr. Then remove the BSA solution, and apply samples and incubate for 2 hr.</li>
		<li>Wash off the samples using 3% BSA in PBS three times, and then add detection antibodies at a concentration provided by the product datasheet. Incubate for 2 hr.</li>
		<li>Wash off the detection antibodies by 3% BSA in PBS three times, and then add Cyanine 5(Cy5)-streptavidin at 1 &#181;g/ml, followed by incubation for 1 hr.</li>
		<li>Repeat step (3.2.4) to clean and dry the slide for scanning.</li>
	</ol>
	</li>
</ol>

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The authors have no competing interests to disclose.

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.

<table><tbody><tr><td>Sylgard 184 silicone elastomer base</td><td>Dow Corning</td><td>3097366-1004</td><td></td></tr><tr><td>Sylgard 184 silicone elastomer curing agent</td><td>Dow Corning</td><td>3097358-1004</td><td></td></tr><tr><td>SU-8 2025 photoresist</td><td>MicroChem</td><td>Y111069</td><td></td></tr><tr><td>Silicon wafers&amp;nbsp;</td><td>University Wafers</td><td>452</td><td></td></tr><tr><td>Poly-L-lysine coated glass slides</td><td>Thermo Scientific</td><td>C40-5257-M20</td><td></td></tr><tr><td>Oligonucleotides</td><td>Integrated DNA Technologies</td><td></td><td>*Custom-ordered from Integrated DNA Technologies, see &lt;strong&gt;Table 2&lt;/strong&gt;.</td></tr><tr><td>Poly-L-lysine adhesive stock solution</td><td>Newcomer Supply</td><td>1339</td><td></td></tr><tr><td>Bis (sulfosuccinimidyl) suberate&amp;nbsp; (BS3)</td><td>Thermo Scientific</td><td>21585</td><td></td></tr><tr><td>1x Phosphate buffered saline, pH 7.4</td><td>Quality Biological</td><td>114-058-101</td><td></td></tr><tr><td>&amp;Auml;kta Explorer 100 Fast Protein Liquid Chromatography (FPLC) System</td><td>GE (Amersham Pharmacia)&amp;nbsp;</td><td>18-1112-41</td><td></td></tr><tr><td>Superose 6 10/300 GL column</td><td>GE Healthcare Life Sciences</td><td>17-5172-01</td><td></td></tr><tr><td>Capture antibodies</td><td>various</td><td>various</td><td>*Antibody selection depends on application</td></tr><tr><td>Succinimidyl-6-hydrazino-nicotinamide (S-HyNic)</td><td>Solulink</td><td>S-1002</td><td></td></tr><tr><td>Succinimidyl-4-formylbenzamide (S-4FB)</td><td>Solulink</td><td>S-1004</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-dimethylformamide</td><td>Sigma-Aldrich</td><td>227056</td><td></td></tr><tr><td>Citric acid, anhydrous</td><td>Acros</td><td>42356</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Fisher Scientific</td><td>S318</td><td></td></tr><tr><td>Amicon Ultra spin filter 10 kDa MWCO</td><td>EMD Millipore</td><td>UFC201024</td><td></td></tr><tr><td>Spin coater</td><td>Laurell Technologies</td><td>WS-650-MZ</td><td></td></tr><tr><td>Biopsy punch with plunger (1.0 mm diameter)</td><td>Ted Pella, Inc.</td><td>15110-10</td><td></td></tr><tr><td>Diamond scribe (Style 60)</td><td>SPI supplies</td><td>6004</td><td></td></tr><tr><td>Trimethylchlorosilane</td><td>Sigma Aldrich</td><td>92361</td><td></td></tr></tbody></table>

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 ...

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Research

JoVE Journal

Chemistry

11.3K Views.  University at Albany, State University of New York. 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.

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

Counting Proteins in Single Cells with Addressable Droplet Microarrays

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behavior within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumor suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10 nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.

Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average ...

Cancer Research

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

Environment

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid&#183;ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of &#946;-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification.

In this article, we present and discuss new developments in the synthesis and applications of nucleic acid microarrays fabricated in situ. Specifically, we show how the protocols for DNA synthesis can be extended to RNA and how microarrays can be used to create retrievable nucleic acid libraries.

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite ...

Genetics

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded&#160;tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image&#160;analysis technique.

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Biology

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).

Fabrication and Use of MicroEnvironment microArrays MEArrays

A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens.
This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera.
Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

We present a protocol for using a protein microarray constructed by printing influenza antigens onto nitrocellulose-coated slides to simultaneously probe serum for multiple antibody isotypes against over 250 antigens from different virus strains, thus allowing measurement of the breadth of serum antibodies across virus subtypes.

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge ...

Immunology and Infection

Detecting Circulating Anti-Glycan Antibodies with a Printed Glycan Array

Source: Olivera-Ardid, S. et al., <a href="https://app.jove.com/v/57662">Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates printed glycan array technology for analyzing circulating anti-glycan antibodies in mouse serum. The antibodies in the serum interact with specific glycans on the chip, which is confirmed by immunostaining followed by fluorescence scanning.

Wykrywanie krążących przeciwciał anty-glikanowych za pomocą drukowanej matrycy glikanów

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Glycochips Production

	...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

A Peptide Microarray Technology for Specificity Profiling of Antibodies

Source: Cornett, E. M., et al. <a href="https://app.jove.com/v/55912">Analysis of Histone Antibody Specificity with Peptide Microarrays.</a> J. Vis. Exp. (2017).
This video demonstrates a technique that applies peptide microarray technology to profile the specificity of antibodies recognizing post-translational modifications (PTMs) on histone peptides. A microarray slide containing immobilized fluorophore-conjugated histone peptides with known PTM combinations is treated with target PTM-specific antibodies. This process helps identify the antibody&#39;s specificity for the target and its potential cross-reactivity with non-target PTMs.

Technologia mikromacierzy peptydowych do profilowania swoistości przeciwciał

1. Designing the Array Slide and Source Plate Layout

	Under the &#39;Plan a slide layout&#39; heading on the ArrayNinja interface, click on the link that ...

A Technique to Detect Cyanobacterial Strains Using an Antibody Microarray Chip

Source: Blanco, Y. et al., <a href="https://app.jove.com/v/54994">Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates a method to detect the Cyanobacterial strains in an environmental sample using an antibody microarray chip. Samples carrying various bacteria interact with printed antibodies, and immunostaining with fluorophore-labeled anti-cyanobacterial-strain antibodies reveals the presence of specific cyanobacterial strains through fluorescence in distinct spots.

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

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Rozdziały w tym wideo

Counting Proteins in Single Cells with Addressable Droplet Microarrays

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

Wykrywanie krążących przeciwciał anty-glikanowych za pomocą drukowanej matrycy glikanów

Technologia mikromacierzy peptydowych do profilowania swoistości przeciwciał

Technika wykrywania szczepów sinic za pomocą chipa mikromacierzy przeciwciał