Name: a guided materials screening approach for developing quantitative sol-gel derived protein microarrays
Uploaded: 2013-08-26T12:00:00
Duration: 10 min 44 s
Description: Watch this Scientific Journal Video about A Guided Materials Screening Approach for Developing Quantitative Sol-gel Derived Protein Microarrays at JoVE.com

The authors thank Maria Monton, Julie Lebert, Jessamyn Little, Xin Ge and Laura Lautens for assistance in development of protein microarrays. The authors also thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for funding this work. The authors also thank the Canada Foundation for Innovation and the Ontario Innovation Trust for support of this work. J.D.B holds the Canada Research Chair in Bioanalytical Chemistry and Biointerfaces.

<ol>
	<li>Schena, M. M., Shalon, D. D., Davis, R. W. R., Brown, P. O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+monitoring+of+gene+expression+patterns+with+a+complementary+DNA+microarray.">Quantitative monitoring of gene expression patterns with a complementary DNA microarray.</a> Science (New York, N.Y.). 270 (5235), 467-470 (1995).</li><li>MacBeath, G., Schreiber, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Printing+proteins+as+microarrays+for+high-throughput+function+determination.">Printing proteins as microarrays for high-throughput function determination.</a> Science (New York, N.Y.). 289 (5485), 1760-1763 (2000).</li><li>Zhu, H. H., Bilgin, M. 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=Global+analysis+of+protein+activities+using+proteome+chips.">Global analysis of protein activities using proteome chips.</a> Science (New York, N.Y.). 293 (5537), 2101-2105 (2001).</li><li>Hook, A. L., Chang, C. -. 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=Combinatorial+discovery+of+polymers+resistant+to+bacterial+attachment.">Combinatorial discovery of polymers resistant to bacterial attachment.</a> Nature Biotechnology. , (2012).</li><li>Monton, M. R. N., Lebert, J. M., Little, J. R. L., Nair, J. J., McNulty, J., Brennan, 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=A+Sol-Gel-Derived+Acetylcholinesterase+Microarray+for+Nanovolume+Small-Molecule+Screening.">A Sol-Gel-Derived Acetylcholinesterase Microarray for Nanovolume Small-Molecule Screening.</a> Analytical Chemistry. 82 (22), 9365-9373 (2010).</li><li>Cho, E. J., Tao, Z., Tehan, E. C., Bright, F. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multianalyte+pin-printed+biosensor+arrays+based+on+protein-doped+xerogels.">Multianalyte pin-printed biosensor arrays based on protein-doped xerogels.</a> Analytical Chemistry. 74 (24), 6177-6184 (2002).</li><li>Cho, E. J., Tao, Z., 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=Tools+to+Rapidly+Produce+and+Screen+Biodegradable+Polymer+and+Sol-Gel-Derived+Xerogel+Formulations.+Applied+Spectroscopy.">Tools to Rapidly Produce and Screen Biodegradable Polymer and Sol-Gel-Derived Xerogel Formulations. Applied Spectroscopy.</a> Society for Applied Spectroscopy. 56 (11), 1385-1389 (2002).</li><li>Ge, X., Lebert, J. M., Monton, M. R. N., Lautens, L. L., Brennan, 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=Materials+Screening+for+Sol-Gel-Derived+High-Density+Multi-Kinase+Microarrays.">Materials Screening for Sol-Gel-Derived High-Density Multi-Kinase Microarrays.</a> Chemistry of Materials. 23 (16), 3685-3691 (2011).</li><li>Rupcich, N., Green, J. R. A., Brennan, 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=Nanovolume+Kinase+Inhibition+Assay+Using+a+Sol-Gel-Derived+Multicomponent+Microarray.">Nanovolume Kinase Inhibition Assay Using a Sol-Gel-Derived Multicomponent Microarray.</a> Analytical Chemistry. 77 (24), 8013-8019 (2005).</li><li>Rupcich, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupled+enzyme+reaction+microarrays+based+on+pin-printing+of+sol-gel+derived+biomaterials.">Coupled enzyme reaction microarrays based on pin-printing of sol-gel derived biomaterials.</a> Analytica Chimica Acta. 500 (1-2), 3-12 (2003).</li><li>MacBeath, G. G., Schreiber, S. L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Printing+proteins+as+microarrays+for+high-throughput+function+determination.">Printing proteins as microarrays for high-throughput function determination.</a> Science (New York, N.Y.). 289 (5485), 1760-1763 (2000).</li><li>Stillman, B. A. B., Tonkinson, J. L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FAST+slides:+a+novel+surface+for+microarrays.">FAST slides: a novel surface for microarrays.</a> BioTechniques. 29 (3), 630-635 (2000).</li><li>Rupcich, N., Goldstein, A., Brennan, 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=Optimization+of+sol-gel+formulations+and+surface+treatments+for+the+development+of+pin-printed+protein+microarrays.">Optimization of sol-gel formulations and surface treatments for the development of pin-printed protein microarrays.</a> Chemistry of Materials. 15 (9), 1803-1811 (2003).</li><li>Gill, I., Ballesteros, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+biologicals+within+silicate,+siloxane,+and+hybrid+sol-gel+polymers:+An+efficient+and+generic+approach.">Encapsulation of biologicals within silicate, siloxane, and hybrid sol-gel polymers: An efficient and generic approach.</a> Journal of the American Chemical Society. 120 (34), 8587-8598 (1998).</li><li>Brook, M. A., Chen, 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=Proteins+entrapped+in+silica+monoliths+prepared+from+glyceroxysilanes.">Proteins entrapped in silica monoliths prepared from glyceroxysilanes.</a> Journal of Sol-gel Science and Technology. 31 (1), 343-348 (2004).</li><li>Brook, M. A., Chen, Y., Guo, K., Zhang, Z., Brennan, 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=Sugar-modified+silanes:+precursors+for+silica+monoliths.">Sugar-modified silanes: precursors for silica monoliths.</a> Journal of Materials Chemistry. 14 (9), 1469 (2004).</li><li>Zhang, J., Chung, T., Oldenburg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> Journal of Biomolecular Screening. 4 (2), 67-73 (1999).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next+generation+of+protein+microarray+support+materials:+Evaluation+for+protein+and+antibody+microarray+applications.">Next generation of protein microarray support materials: Evaluation for protein and antibody microarray applications.</a> Journal of Chromatography A. , 1-8 (2013).</li><li>Bhatia, R. B., Brinker, C. J., Gupta, A. K., Singh, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+Sol-Gel+Process+for+Protein+Encapsulation.">Aqueous Sol-Gel Process for Protein Encapsulation.</a> Chemistry of Materials. 12 (8), 2434-2441 (2000).</li></ol>

We have nothing to disclose.

The methodology described here was selected as the most suitable for identifying printable sol-gel derived materials with a contact printer, producing a time- and cost-effective procedure for rapidly identifying optimal materials without having to screen large numbers of materials. From a total of ~20,000 potential materials, it was possible to identify ~200 materials that were suitable for printing on the basis of gelation time alone. This significantly reduced the number of materials needed to be prepared for subsequent printing trials. These printable materials were then printed onto 4 slide surfaces for a total of 768 material-slide combinations. On average, 50 spots/replicates of one material can be printed in ~3 min, including sample loading, spot deposition and pin cleaning. Of those, 155 materials, or roughly 20%, allowed for printing the maximum number of spots per solution uptake and produced reproducible spot sizes. It should be noted that of the 4 slide surfaces tested, materials printed better in the order: amine &#62; epoxy &#62; aldehyde &#62; PMMA; PMMA slides did not produce useful arrays for any materials. This was likely attributed to the polarity of the surface coating. Comparing the aforementioned slide surfaces, the more polar amine and epoxy were better suited for the aqueous sols compared to the PMMA slides. Furthermore, of the tested surfaces, the amine coated slides provide a potential positively charged surface for the deposited anionic sol to bond. We suspect, the silica nanoparticles at the interface between the slide and the sol interact along the surface. Both the epoxy and the aldehyde slide surfaces lack the same initial charge-based interaction. To ensure optimal spot deposition it is highly recommended to use pre-coated slides from a supplier such as Arrayit. In-house coating produces inconsistent surfaces that lead to poor spot reproducibility13 and, in some cases, may lead to quantification problems.18 Of equal importance, temperature and humidity affect the &#34;printability&#34; of the materials. While no detailed studies on the effects related to temperature were carried out, printing was always carried out at room temperature (23&#177;3 &#176;C). Humidity (greater than 80%) was also controlled within the print chamber to prevent irregular shape deposition due to small deposition volumes (0.7-2.3 nl) and evaporation.
While the material screen was guided towards identifying optimal sol-gel derived materials specifically for printing of AChE and kinases, a small set of materials were identified that worked for both types of proteins. Indeed, both of the materials that were identified for kinase microarray fabrication were based on SS+PVA+glycerol, and both materials were also identified within the 26 materials selected for AChE microarrays. These &#34;optimal&#34; materials may offer a generic starting point for developing further protein-doped sol-gel based microarrays, and small screens centered around these compositions may identify even better materials for microarray fabrication. A second point to note is the importance of the enzyme used. In the case of AChE (a rather robust enzyme), 26 (or roughly 40%) of the original 66 materials identified as assay-compatible retained the activity of entrapped AChE. However, for the more delicate kinases, only 2 of the 69 assay-compatible compositions, or roughly 3% of the materials, were able to retain the activity of all kinases. While sufficient numbers of different enzymes have not been studied to make conclusive statements, it appears that optimizing array fabrication with relatively unstable enzymes may lead to identification of materials that can entrap a wide range of proteins to allow mutliplexed microarray fabrication.
Independent of the chosen protein, the major cut-off factor for identifying printable materials was the need for a long material gelation times (&#62;2.5 hr). When developing SS based sol-gel materials, it is very important to ensure that, following ion exchange and filtration, the sol is at about pH 4. Sols with a lower initial pH may result in materials with a lower-than-neutral pH, which can affect enzyme activity.19 Adjusting the amount of Dowex (ion exchange resin) to SS can alter the final pH of the sol. When a new batch of the resin is prepared the ratio of resin to SS needs to be adjusted so as to produce sols at about pH 4 following the procedure in section 2 of the protocol.
Similarly, the preparation of crystalline DGS is often a source of error associated with material failure when using DGS based sols for the biomolecule entrapment. Although not reported here in detail, great care needs to be taken during the synthesis of crystalline DGS, in particular the need to avoid the presence of water during the synthesis, which can produce polyglycerated silicates rather than monomeric DGS. Also, due to the hygroscopic nature of DGS, the crystalline sample needs to be stored desiccated and used within 6 months after synthesis. Crystalline DGS older than 6 months may not dissolve fully (owing to partially condensed polyglyceryl silicate material) even with sonication in an acidic environment. Incomplete DGS dissolution produces sols with unknown and uncontrollable silica content and thus, less robust materials.
An important point to note with contact printing is the quality of the pins. Damaged or mishandled pins (Figure 9) will never produce reproducible arrays independent of the material being printed. It is recommended to check pin quality using a dissection microscope to ensure broken or clogged pins are not used. Careful handling ensures long life for the pins. Free movement of the pin is also important. In cases where moisture is trapped in the print head between the head and the pin, the pin will not seat correctly and thus will not make proper contact with the surface, resulting in a lack of deposition of material.
In conclusion, we have provided a detailed, multistep screening approach for developing high-density protein doped pin-printed microarrays. The screening involves optimization of material properties (gelation time and printability) to allow printing of materials, followed by more focused screening to identify materials that are compatible with a given assay and able to retain enzyme activity. This guided material screening approach can be applied to additional microarray formats to reduce time and cost associated with producing efficient high-density microarrays.

Microarrays have gained popularity within the scientific community as a method for increasing assay throughput. Since the development of microarray technology to assess gene expression1 in the mid-1990s, microarrays have found use in the development of high-throughput assays to identify protein-protein and protein-small molecule interactions, and to find new materials with unique properties.2-5 More recently, microarrays have been developed wherein specific materials are used to immobilize functional proteins, producing a three-dimensional microarray element within which proteins are entrapped, allowing for facile measurement of enzymatic activity and inhibition on the microarray itself using a suitable fluorescence assay that is coupled to substrate turnover.5-10 Importantly, such microarrays can be designed to include all necessary components to screen samples and controls together in a highly paralleled fashion.5,8
Early examples of protein microarrays were typically prepared using standard methods for protein immobilization onto solid supports, such as covalent attachment,11 affinity capture3 and physical adsorption.12 While these methods of bio-immobilization allow for increased sample concentration and accelerated reaction kinetics required for assay miniaturization, they each suffer drawbacks. In general, all cause a reduction in native biomolecule functionality owing to chemical modification of the surface, hindered access to active sites, or improper orientation due to non-specific immobilization. Thus, all methods result in lower assay sensitivity despite an increase in biomolecule deposition, a response that likely arises due to the need to artificially bind biomolecules to a surface.
An emerging approach for the production of functional biomolecule microarrays is through pin-printing of protein-doped silica sols onto solid supports, which are typically either functionalized glass slides or individual wells of microwell plates. The sol-gel process itself takes place in an aqueous environment at room temperature, and it is the liquid precursor that is printed and then gels to entrap biomolecules within the 3D matrix, allowing for high protein loading13 as well as entrapment of multiple components within the same microarray element.13,14 Tailoring of the sol-gel derived material can be done through careful selection of different silica precursors, as well as by altering the aqueous component through use of different buffers (pH, ionic strength), and inclusion of various additives (polymers, small molecules) to achieve an optimal material, the specific nature of which depends on the biomolecule that is entrapped.10
A potential limitation associated with developing sol-gel derived protein microarrays via pin-printing is the need to identify sol-gel based composite materials that can be printed without gelling in the pin or showing undesirable properties (irreproducible spot sizes, cracking, poor adhesion, incompatibility with assay components, poor protein activity) once printed on a surface.5 Simultaneous optimization of all of these parameters precludes an approach wherein materials can be designed de novo, or examined slowly in a serial manner. On the other hand, random screening of many thousand or tens of thousands of materials is neither time- nor cost-effective.
In this article, we describe a directed screening approach that allows rapid identification of suitable materials for production of protein microarrays without the need to randomly screen large numbers of materials. Using a guided approach, materials suitable for microarray printing are first identified, followed by a series of small-scale screens to identify optimal sol-gel derived material combinations that can be printed reproducibly, without cracking and are compatible with a given assay. Finally, optimal materials are identified based on retention of enzyme activity and performance in a final small-molecule screening assay. In this way, optimal materials can be identified from many thousand candidates using only a few hundred assay steps. We demonstrate this approach for fabrication of both high-density acetylcholinesterase and multikinase microarrays and the use of such microarrays for small-molecule screening.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent/Material</td>
		</tr>
		<tr>
			<td>Poly(vinyl alcohol) (PVA)</td>
			<td>Sigma-Alderich</td>
			<td>360627</td>
			<td>80% hydrolozyed, Mw 600</td>
		</tr>
		<tr>
			<td>Polyethylene glycol 600 (PEG)</td>
			<td>Sigma-Alderich</td>
			<td>87333</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine (PEI)</td>
			<td>Sigma-Alderich</td>
			<td>482595</td>
			<td>50% (w/w) solution in water</td>
		</tr>
		<tr>
			<td>Carboxyethylsilanetriol (Si-COOH)</td>
			<td>Gelest, Inc.</td>
			<td>SIC2263.0</td>
			<td>25% in water</td>
		</tr>
		<tr>
			<td>N-(3-triethoxysilylpropyl) gluconamide(GLS)</td>
			<td>Gelest, Inc.</td>
			<td>SIT8189.0</td>
			<td>50% in ethanol</td>
		</tr>
		<tr>
			<td>bis[(3-methyldimethoxysilyl)propyl]polypropylene oxide (MDSPPO)</td>
			<td>Gelest, Inc.</td>
			<td>SIB1660.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyltrimethoxysilane (MTMS)</td>
			<td>Gelest, Inc.</td>
			<td>SIM6560.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Bis(triethoxysiyly)ethane (Bis-TEOS)</td>
			<td>Gelest, Inc.</td>
			<td>SIB1817.0</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Aminopropyltriethoxysilane (APTES)</td>
			<td>Gelest, Inc.</td>
			<td>SIA0610.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Alderich</td>
			<td>49767</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Sorbitol</td>
			<td>Sigma-Alderich</td>
			<td>240850</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Trehalose dihydrate</td>
			<td>Sigma-Alderich</td>
			<td>T9531</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Alderich</td>
			<td>X-100</td>
			<td></td>
		</tr>
		<tr>
			<td>N&#949;-Acetyl-L-lysine</td>
			<td>Sigma-Alderich</td>
			<td>A4021</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Sigma-Alderich</td>
			<td>154563</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Alderich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide, 1.0 N</td>
			<td>LabChem Inc.</td>
			<td>LC24350-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid, 1.0 N/0.1 N</td>
			<td>LabChem Inc.</td>
			<td>LC15300-2/LC152220-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Alderich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Diglycerolsilane (DGS)</td>
			<td></td>
			<td></td>
			<td>Prepared in laboratory</td>
		</tr>
		<tr>
			<td>Sodium silicate solution</td>
			<td>Fisher Scientific</td>
			<td>SS338-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dowex 50WX8-100 ion exchance resin</td>
			<td>Sigma-Alderich</td>
			<td>217492</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetylthiocholine iodide</td>
			<td>Sigma-Alderich</td>
			<td>1480</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetylcholinesterase from Electrophorus electricus (electric eel)</td>
			<td>Sigma-Alderich</td>
			<td>C2888</td>
			<td></td>
		</tr>
		<tr>
			<td>BODIPY FL L-Cystine</td>
			<td>Invitrogen</td>
			<td>B-20340</td>
			<td></td>
		</tr>
		<tr>
			<td>Pro-Q Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit</td>
			<td>Invitrogen</td>
			<td>P33706</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#39;triphosphate disodium salt (ATP) solution</td>
			<td>Sigma-Alderich</td>
			<td>A6559</td>
			<td></td>
		</tr>
		<tr>
			<td>MAP Kinase 2 (MAPK2)</td>
			<td>EMD Millipore</td>
			<td>454850</td>
			<td></td>
		</tr>
		<tr>
			<td>p38&#945;/SAPK2a (T106M), active</td>
			<td>EMD Millipore</td>
			<td>14-687M</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal growth factor (EGFR)</td>
			<td>EMD Millipore</td>
			<td></td>
			<td>Donated by Millipore</td>
		</tr>
		<tr>
			<td>Glycogen synthase kinase 3&#946; (GSK-3&#946;)</td>
			<td>EMD Millipore</td>
			<td>14-306</td>
			<td></td>
		</tr>
		<tr>
			<td>Myelin basic protein (MBP)</td>
			<td>EMD Millipore</td>
			<td></td>
			<td>Substrate for MAPK2 and p38&#945;, Donated by Millipore</td>
		</tr>
		<tr>
			<td>GSM</td>
			<td>EMD Millipore</td>
			<td>12-533</td>
			<td>Substrate for GSK-3&#946;</td>
		</tr>
		<tr>
			<td>Poly-glu-tyr polypeptide p(E4Y)</td>
			<td>EMD Millipore</td>
			<td>12-440</td>
			<td>Substrate for EGFR</td>
		</tr>
		<tr>
			<td>Stealth pin</td>
			<td>ArrayIt</td>
			<td>SMP3</td>
			<td></td>
		</tr>
		<tr>
			<td>Stealth pin</td>
			<td>ArrayIt</td>
			<td>SMP7</td>
			<td></td>
		</tr>
		<tr>
			<td>Amine coated slides</td>
			<td>ArrayIt</td>
			<td>SMM2</td>
			<td></td>
		</tr>
		<tr>
			<td>Aldehyde coated slides</td>
			<td>ArrayIt</td>
			<td>SMA2</td>
			<td></td>
		</tr>
		<tr>
			<td>Exposy coated slides</td>
			<td>ArrayIt</td>
			<td>SME2</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(methylmethacrylate) (PMMA) coated slides</td>
			<td>Exakt Technologies Inc.</td>
			<td>41500</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2-&#956;m syringe filter</td>
			<td>PALL Life Sciences</td>
			<td>4612</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Virtek Contact Printer</td>
			<td>BioRad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Novaray Fluorescence Slide Imager</td>
			<td>Alpha Innotech Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop microarray centrifuge</td>
			<td>ArrayIt</td>
			<td>MHC110V</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliQ Synthesis A10</td>
			<td>Millipore</td>
			<td></td>
			<td>Used to filter all water required for experiments</td>
		</tr>
	</tbody>
</table>

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.

By performing a guided factor analysis for the material screen, we were able to minimize the number of materials tested from ~20,000 to a few hundred that had gelation times suitable for printing. By applying a strict guideline requiring material gelation time of 2.5 hr or greater, materials likely to clog the printing pins or produce irreproducible arrays were never printed. The printable materials identified to have sufficient (&#62;2.5 hr) gelation times were printed onto 4 different functionalized glass slide surfaces. In order to be considered &#34;printable&#34;, the maximum number of spots per uptake volume of the pin had to be printed (SMP3 = 200). Spots were also assessed for spot morphology to ensure no cracking or undesirable phase separation had occurred using simple brightfield microscopy as shown in Figure 2.
From this stage of identified printable materials, microarrays were produced with AChE and kinases incorporated into the buffered aqueous component. Materials that were compatible with the assay procedure (including potential overprinting and washing or staining steps) were identified by observing retention of microarray spots (no cracking, loss of spots or unusual fluorescence patterns) and a positive control (PC) to negative control (NC) ratio greater than 1 as observed through image. As this was roughly 50% of the materials, a greater PC/NC ratio of 3 was used to define optimal materials with retention of protein activity. Through this method, 26 sol-gel derived materials containing AChE and 2 materials containing kinases satisfied the &#62;3 PC/NC criteria. Figure 3 and Figure 4 show a graphical breakdown of the 5 guided material screen steps for the identification of optimal AChE and kinase microarrays, respectively.
The assays could also be validated through the generation of a Z&#39; score.17 This was done using the material that produced the highest PC/NC ratio. Figure 5 shows the Z&#39; plot obtained by comparing the signal generated from 200 spots, 100 PC and 100 NC after overprinting the indicator dye and substrate on the AChE array. The AChE and the kinase arrays resulted in the respective Z&#39; scores of 0.60 and 0.67, indicative of an excellent assay. However, it should be noted that before assay validation, on-array enzyme, dye, substrate and cofactor concentrations had to be optimized by overprinting a range of concentrations of each component and selecting the concentration that produced the highest signal, as described in detail elsewhere.5
To validate the assays, quantitative inhibition data were obtained using known and unknown AChE inhibitors, with results performed in duplicate and used to produce duplicate plots (Figure 6A) and inhibition curves (Figures 6B and 6C). Spots were first overprinted with mixtures of known biologically active small-molecule inhibitors then with dye and substrate, and control mixtures containing either known inhibitors or no inhibitor were included. Duplicate plots were generated to assess enzyme activity, and any mixtures that resulted in less than 25% enzyme activity were considered positive for inhibition. Individual compounds from such mixtures were then tested in duplicate to identify the specific small molecule(s) responsible for inhibition. Once identified, these small molecules were used to generate quantitative inhibition curves to determine IC50 values and inhibition constants.
Similar qualitative results were obtained using the multikinase array with a common kinase inhibitor, staurosporine. Figure 7A and 7B show the microarray image and indicate that the signal intensities after overprinting and staining the multikinase array are as expected for a negative control (- ATP), positive control (+ ATP) and known inhibitor (+ ATP + inh). To demonstrate the ability to obtain quantitative inhibition data from the microarrays, a concentration dependent inhibition assay was done for a single kinase. As shown in Figures 8A and 8B, signal intensity decreases as inhibitor concentration increases, and the response follows the expected concentration dependent inhibition curve for the p38&#945;/MBP kinase/substrate system.
<img alt="Figure 1" src="/files/ftp_upload/50689/50689fig1.jpg" /> 
Figure 1. General schematic for the guided materials screening approach. Each block represents a step of the screen in sequential order. Numbers on the left represent the total number of materials prepared for analysis. Using a gelation time greater than 2.5 hr (materials with gelation times less than 2.5 hr are indicated by the strikeout), the number of materials that passed each stage and carried forward during the material screen are indicated by the number on the right. *Represents materials with less than optimal phase separation.
<img alt="Figure 2" src="/files/ftp_upload/50689/50689fig2.jpg" /> 
Figure 2. Optical images showing various failure modes of materials at the printability step of the screen. An image of a &#34;good&#34; material (second row, third column) is also shown for comparison. Reprinted with permission from reference 8, copyright 2013 American Chemical Society.
<img alt="Figure 3" src="/files/ftp_upload/50689/50689fig3.jpg" /> 
Figure 3. A directed material screening approach for identification of optimal materials for fabricating sol-gel-derived AChE microarrays. Reprinted with permission from reference 5, copyright 2013 American Chemical Society.
<img alt="Figure 4" src="/files/ftp_upload/50689/50689fig4.jpg" /> 
Figure 4. A directed materials screening approach for identification of optimal materials for fabricating sol- gel-derived kinase microarrays. Reprinted with permission from reference 8, copyright 2013 American Chemical Society.
<img alt="Figure 5" src="/files/ftp_upload/50689/50689fig5.jpg" /> 
Figure 5. (A) A section of AChE microarray showing HC (bright green) and LC (light green) spots (a black-green palette was applied as pseudocolor for clarity of presentation); (B) a magnified view of the boxed area to highlight spot morphology and alignment; and (C) a Z&#39; plot. Solid lines indicate the mean of the replicates, while dashed lines correspond to 3SD. Reprinted with permission from reference 5, copyright 2013 American Chemical Society.
<img alt="Figure 6" src="/files/ftp_upload/50689/50689fig6.jpg" /> 
Figure 6. (A) Duplicate plot for on-array screening of synthetic analogs of Amaryllidaceae alkaloids; (B) IC50 plots of identified potential inhibitors marked as compounds 1 and (C) compound 2, with error bars representing one standard deviation of the mean from 25 replicates. Representative spots are shown to illustrate differences in signal proportional to inhibitor concentrations. Reprinted with permission from reference 5, copyright 2013 American Chemical Society. <a href="https://www.jove.com/files/ftp_upload/50689/50689fig6large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 7" src="/files/ftp_upload/50689/50689fig7.jpg" /> 
Figure 7. On-array assay of four kinases using 1.4SS/1.0PVA for entrapment and printed onto an amine-derivatized slide. (A) An image of a section of microarray in which spots with kinases co-entrapped with their respective substrates were overprinted with buffer (NC, top row), or solutions containing ATP (PC, middle row) or ATP + staurosporine (bottom row). (B) Bar graphs comparing signal intensities between inhibited and uninhibited reactions, after subtraction of background signals and error bars representing one standard deviation of the mean from 25 replicates. Reprinted with permission from reference 8, copyright 2013 American Chemical Society.
<img alt="Figure 8" src="/files/ftp_upload/50689/50689fig8.jpg" /> 
Figure 8. Inhibition assay on a p38a/MBP microarray. (A) Sections of microarrays showing representative spots overspotted with varying concentrations of staurosporine, as indicated (the images were obtained by a single scan of the same slide; composite image is shown for clarity). (B) IC50 curve generated from the analyzed array images. The intensity obtained at 100 mM was subtracted from all images; all other intensities were normalized by setting the intensity obtained at 10 nM to a value of 100% activity. Reprinted with permission from reference 8, copyright 2013 American Chemical Society.
<img alt="Figure 9" src="/files/ftp_upload/50689/50689fig9.jpg" /> 
Figure 9. Images of microscopic stealth pin used for contact pin-printing showing various imperfections: (A) clogged, (B) bent.

Watch this Scientific Journal Video about A Guided Materials Screening Approach for Developing Quantitative Sol-gel Derived Protein Microarrays at JoVE.com

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

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