Name: Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray (Video) | JoVE Journal
Uploaded: 2016-01-06T15:00:01.000+00:00
Duration: 9 min 5 s
Description: Learn flow-pattern guided fabrication of high-density barcode antibody microarrays for single-cell protein detection. This customizable method enables simultaneous analysis of secret and intracellular proteins.

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5 minutes

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

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...

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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Research

JoVE Journal

Chemistry

We described a procedure for the disaggregation of colorectal cancer (CRC) to produce viable single cells, which are then captured on customized antibody microarrays recognizing surface antigens (DotScan CRC microarray).  Sub-populations of cells bound to the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with fluorescent dyes.

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Polymer Microarrays for High Throughput Discovery of Biomaterials

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.

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

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.

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

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.

Detecting Circulating Anti-Glycan Antibodies with a Printed Glycan Array

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's specificity for the target and its potential cross-reactivity with non-target PTMs.

A Peptide Microarray Technology for Specificity Profiling of Antibodies

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.

A Technique to Detect Cyanobacterial Strains Using 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.

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

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.

Counting Proteins in Single Cells with Addressable Droplet Microarrays

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.

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

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 analysis technique.

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

Learn flow-pattern guided fabrication of high-density barcode antibody microarrays for single-cell protein detection. This customizable method enables simultaneous analysis of secret and intracellular proteins.

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

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