Name: flow-pattern guided fabrication of high-density barcode antibody microarray
Uploaded: 2016-01-06T15:00:01
Description: Watch this Scientific Journal Video about Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray at JoVE.com

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>
	<li>Alhamdani, M. S. S., Schroder, C., Hoheisel, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncoproteomic+profiling+with+antibody+microarrays.">Oncoproteomic profiling with antibody microarrays.</a> Genome Med. 1 (7), (2009).</li><li>Angenendt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+protein+and+antibody+microarray+technology.">Progress in protein and antibody microarray technology.</a> Drug Discov Today. 10 (7), 503-511 (2005).</li><li>Sun, H. Y., Chen, G. Y. J., Yao, S. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+microarray+technologies+for+proteomics.">Recent advances in microarray technologies for proteomics.</a> Chem Biol. 20 (5), 685-699 (2013).</li><li>Fan, R., 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=Integrated+barcode+chips+for+rapid,+multiplexed+analysis+of+proteins+in+microliter+quantities+of+blood.">Integrated barcode chips for rapid, multiplexed analysis of proteins in microliter quantities of blood.</a> Nat Biotechnol. 26 (12), 1373-1378 (2008).</li><li>Chattopadhyay, P. K., Gierahn, T. M., Roederer, M., Love, J. 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. D., Heath, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma+cellular+architectures+are+predicted+through+the+characterization+of+two-cell+interactions.">Glioblastoma cellular architectures are predicted through the characterization of two-cell interactions.</a> Proc Natl Acad Sci USA. 111 (17), 6521-6526 (2014).</li><li>Xue, M., 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=Chemical+methods+for+the+simultaneous+quantitation+of+metabolites+and+proteins+from+single+cells.">Chemical methods for the simultaneous quantitation of metabolites and proteins from single cells.</a> J Am Chem Soc. 137 (12), 4066-4069 (2015).</li><li>Shi, Q. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-encoded+antibody+libraries:+A+unified+platform+for+multiplexed+cell+sorting+and+detection+of+genes+and+proteins.">DNA-encoded antibody libraries: A unified platform for multiplexed cell sorting and detection of genes and proteins.</a> J Am Chem Soc. 129, 1959-1967 (2007).</li><li>Alegria-Schaffer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+protein-protein+cross-linking.">General protein-protein cross-linking.</a> Methods Enzymol. 539, 81-87 (2014).</li><li>Ahmad, 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=A+robotics+platform+for+automated+batch+fabrication+of+high+density,+microfluidics-based+DNA+microarrays,+with+applications+to+single+cell,+multiplex+assays+of+secreted+proteins.">A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins.</a> Rev Sci Instrum. 82, 094301 (2011).</li></ol>

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 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>
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			<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>
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			<td height="21" style="height:21px;width:263px;">Oligonucleotides</td>
			<td style="width:403px;">Integrated DNA Technologies</td>
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			<td>*Custom-ordered from Integrated DNA Technologies, see table below</td>
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			<td height="21" style="height:21px;width:263px;">1x Phosphate buffered saline, pH 7.4</td>
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			<td height="40" style="height:40px;width:263px;">&#196;kta Explorer 100 Fast Protein Liquid Chromatography (FPLC) System</td>
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			<td>18-1112-41</td>
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			<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>
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			<td height="40" style="height:40px;width:263px;">Succinimidyl-6-hydrazino-nicotinamide (S-HyNic)</td>
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			<td style="width:119px;">S-1002</td>
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			<td style="width:403px;">Solulink</td>
			<td style="width:119px;">S-1004</td>
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			<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>
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			<td height="21" style="height:21px;width:263px;">Citric acid, anhydrous</td>
			<td style="width:403px;">Acros</td>
			<td style="width:119px;">42356</td>
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			<td height="21" style="height:21px;width:263px;">Sodium hydroxide</td>
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			<td style="width:119px;">S318</td>
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			<td style="width:403px;">Sigma Aldrich</td>
			<td align="right" style="width:119px;">92361</td>
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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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Chemistry

A Guided Materials Screening Approach for Developing Quantitative Sol-gel Derived Protein Microarrays

Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a guided material screening approach to identify sol-gel based materials that are suitable for producing three-dimensional protein microarrays. The approach first identifies materials that can be printed as microarrays, narrows down the number of materials by identifying those that are compatible with a given enzyme assay, and then hones in on optimal materials based on retention of maximum enzyme activity. This approach is applied to develop microarrays suitable for two different enzyme assays, one using acetylcholinesterase and the other using a set of four key kinases involved in cancer. In each case, it was possible to produce microarrays that could be used for quantitative small-molecule screening assays and production of dose-dependent inhibitor response curves. Importantly, the ability to screen many materials produced information on the types of materials that best suited both microarray production and retention of enzyme activity. The materials data provide insight into basic material requirements necessary for tailoring optimal, high-density sol-gel derived microarrays.

A guided material screening approach to develop sol-gel derived protein doped microarrays using an emerging pin-printing method of fabrication is described. This methodology is demonstrated through the development of acetylcholinesterase and multikinase microarrays, which are used for cost-effective small-molecule screening.

Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a ...

Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 &#956;m polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 &#956;m microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

We describe a procedure for profiling salivary proteins using multiplexed microsphere-based antibody arrays. Monoclonal antibodies were covalently linked to fluorescent dye-encoded 4.5 &#x03BC;m polymer microspheres using carbodiimide chemistry. The modified microspheres were deposited in fiber-optic microwells to measure protein levels in saliva using fluorescence sandwich immunoassays.

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array ...

Fabrication of Large-area Free-standing Ultrathin Polymer Films

This procedure describes a method for the fabrication of large-area and ultrathin free-standing polymer films. Typically, ultrathin films are prepared using either sacrificial layers, which may damage the film or affect its mechanical properties, or they are made on freshly cleaved mica, a substrate that is difficult to scale. Further, the size of ultrathin film is typically limited to a few square millimeters. In this method, we modify a surface with a polyelectrolyte that alters the strength of adhesion between polymer and deposition substrate. The polyelectrolyte can be shown to remain on the wafer using spectroscopy, and a treated wafer can be used to produce multiple films, indicating that at best minimal amounts of the polyelectrolyte are added to the film. The process has thus far been shown to be limited in scalability only by the size of the coating equipment, and is expected to be readily scalable to industrial processes. In this study, the protocol for making the solutions, preparing the deposition surface, and producing the films is described.

We describe a method for the fabrication of large-area (up to 13 cm diameter) and ultrathin (as thin as 8 nm) polymer films. Instead of using a sacrificial interlayer to delaminate the film from its substrate, we use a self-limiting surface treatment suitable for arbitrarily large areas.

This procedure describes a method for the fabrication of large-area and ultrathin free-standing polymer films.  Typically, ultrathin films are prepared ...

Microbubble Fabrication of Concave-porosity PDMS Beads

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is particularly useful for separations applications. Porous polydimethylsiloxane (PDMS) beads can be produced by heat-curing such an emulsion, allowing the interface between the aqueous and aliphatic phases to mold the morphology of the polymer. In the procedures described here, both polymer and crosslinker (triethoxysilane) are sonicated together in a cold-bath sonicator. Following a period of cross-linking, emulsions are added dropwise to a hot surfactant solution, allowing the aqueous phase of the emulsion to separate, and forming porous polymer beads. We demonstrate that this method can be tuned, and the SAV ratio optimized, by adjusting the electrolyte content of the aqueous phase in the emulsion. Beads produced in this way are imaged with scanning electron microscopy, and representative SAV ratios are determined using Brunauer&#8211;Emmett&#8211;Teller (BET) analysis. Considerable variability with the electrolyte identity is observed, but the general trend is consistent: there is a maximum in SAV obtained at a specific concentration, after which porosity decreases markedly.

Procedures used to generate microstructured concave-porosity polydimethylsiloxane beads are presented. Effects of electrolyte concentration and identity within the aqueous phase are particularly emphasized.

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is ...

Influence of Hybrid Perovskite Fabrication Methods on Film Formation, Electronic Structure, and Solar Cell Performance

Hybrid organic/inorganic halide perovskites have lately been a topic of great interest in the field of solar cell applications, with the potential to achieve device efficiencies exceeding other thin film device technologies. Yet, large variations in device efficiency and basic physical properties are reported. This is due to unintentional variations during film processing, which have not been sufficiently investigated so far. We therefore conducted an extensive study of the morphology and electronic structure of a large number of CH3NH3PbI3 perovskite where we show how the preparation method as well as the mixing ratio of educts methylammonium iodide and lead(II) iodide impact properties like film formation, crystal structure, density of states, energy levels, and ultimately the solar cell performance.

We present an extensive study on the effects of different fabrication methods for organic/inorganic perovskite thin films by comparing crystal structures, density of states, energy levels, and ultimately the solar cell performance.

Hybrid organic/inorganic halide perovskites have lately been a topic of great interest in the field of solar cell applications, with the potential to ...

Fabrication and Testing of Photonic Thermometers

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being leveraged for developing sophisticated photonic sensors. Silicon photonic sensors seek to exploit the strong confinement of light in nano-waveguides to transduce changes in physical state to changes in resonance frequency. In the case of thermometry, the thermo-optic coefficient, i.e., changes in refractive index due to temperature, causes the resonant frequency of the photonic device such as a Bragg grating to drift with temperature. We are developing a suite of photonic devices that leverage recent advances in telecom compatible light sources to fabricate cost-effective photonic temperature sensors, which can be deployed in a wide variety of settings ranging from controlled laboratory conditions, to the noisy environment of a factory floor or a residence. In this manuscript, we detail our protocol for the fabrication and testing of photonic thermometers.

We describe the process of fabrication and testing of photonic thermometers.

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being ...

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise &#945;-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial &#946;-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

Fabrication and Testing of Catalytic Aerogels Prepared Via Rapid Supercritical Extraction

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three preparation methods are described: (a) the incorporation of metal salts into silica or alumina wet gels using an impregnation method; (b) the incorporation of metal salts into alumina wet gels using a co-precursor method; and (c) the addition of metal nanoparticles directly into a silica aerogel precursor mixture. The methods utilize a hydraulic hot press, which allows for rapid (&#60;6 h) supercritical extraction and results in aerogels of low density (0.10 g/mL) and high surface area (200-800 m2/g). While the work presented here focuses on the use of copper salts and copper nanoparticles, the approach can be implemented using other metal salts and nanoparticles. A protocol for testing the three-way catalytic ability of these aerogels for automotive pollution mitigation is also presented. This technique uses custom-built equipment, the Union Catalytic Testbed (UCAT), in which a simulated exhaust mixture is passed over an aerogel sample at a controlled temperature and flow rate. The system is capable of measuring the ability of the catalytic aerogels, under both oxidizing and reducing conditions, to convert CO, NO and unburned hydrocarbons (HCs) to less harmful species (CO2, H2O and N2). Example catalytic results are presented for the aerogels described.

Here we present protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms. Methods for preparing materials using copper salts and copper-containing nanoparticles are featured. Catalytic testing protocols demonstrate the effectiveness of these aerogels for three-way catalysis applications.

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three ...

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands for in vivo investigations. Within this protocol, a robust and reliable remote-controlled radiosynthesis of [11C]SNAP-7941, an antagonist to the melanin-concentrating hormone receptor 1, is described. The radiosynthesis starts with cyclotron produced [11C]CO2 that is subsequently further reacted via a gas-phase transition to [11C]CH3OTf. Then, this reactive intermediate is introduced to the precursor solution and forms the respective radiotracer. Chemical as well as the radiochemical purity are determined by means of RP-HPLC, routinely implemented in the radiopharmaceutical quality control process. Additionally, the molar activity is calculated as it is a necessity for the following real-time kinetic investigations. Furthermore, [11C]SNAP-7941 is applied to MDCKII-WT and MDCKII-hMDR1 cells for evaluating the impact of P-glycoprotein (P-gp) expression on cell accumulation. For this reason, the P-gp expressing cell line (MDCKII-hMDR1) is either used without or with blocking prior to experiments by means of the P-gp substrate (&#177;)-verapamil and the results are compared to the ones observed for the wildtype cells. The overall experimental approach demonstrates the importance of a precise time-management that is essential for every preclinical and clinical study using PET tracers radiolabelled with short-lived nuclides, such as carbon-11 (half-life: 20 min).

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Here, we represent a protocol for the fully automated radiolabelling of [11C]SNAP-7941 and the analysis of the real-time kinetics of this PET-tracer on P-gp expressing and non-expressing cells.

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands ...

Fabrication of Thin Film Silver/Silver Chloride Electrodes with Finely Controlled Single Layer Silver Chloride

This paper aims to present a protocol to form smooth and well-controlled films of silver/silver chloride (Ag/AgCl) with designated coverage on top of thin film silver electrodes. Thin film silver electrodes sized 80 &#181;m x 80 &#181;m and 160 &#181;m x 160 &#181;m were sputtered on quartz wafers with a chromium/gold (Cr/Au) layer for adhesion. After passivation, polishing and cathodic cleaning processes, the electrodes underwent galvanostatic oxidation with consideration of Faraday&#39;s Law of Electrolysis to form smooth layers of AgCl with a designated degree of coverage on top of the silver electrode. This protocol is validated by inspection of scanning electron microscope (SEM) images of the surface of the fabricated Ag/AgCl thin film electrodes, which highlights the functionality and performance of the protocol. Sub-optimally fabricated electrodes are fabricated as well for comparison. This protocol can be widely used to fabricate Ag/AgCl electrodes with specific impedance requirements (e.g., probing electrodes for impedance sensing applications like impedance flow cytometry and interdigitated electrode arrays).

This paper aims to present a method to form smooth and well-controlled films of silver chloride (AgCl) with designated coverage on top of thin film silver electrodes.

This paper aims to present a protocol to form smooth and well-controlled films of silver/silver chloride (Ag/AgCl) with designated coverage on top of thin ...