Hydrogel-Based Protein Nanoarrays

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Abstract:

This paper explores the use of surface-patterned nanohydrogels as a substrate for high-density and high-sensitivity protein arrays. Nanohydrogels were created by locally crosslinking dry amine-terminated PEG 5000 thin films using a focused electron beam. Unirradiated polymer was subsequently washed away leaving behind gels approximately 200 nm in diameter with a dry height of about 50nm which swell in water by a factor of about five. Two different protein assays involving the nucleic acid binding protein zinc finger 9 (ZNF9) were developed which covalently bind reagents to the amine groups within the PEG nanohydrogels. One directly binds ZNF9 while the other binds α-GST antibody to mediate attachment of GST-tagged ZNF9. In both cases 100 m diameter spots containing 7500 discrete nanohydrogels were patterned into a format consistent with equivalent microarrays created by spotting reagents onto four different commercially available substrates. The arrays were interrogated using a fluorescently labeled oligonucleotide known to bind ZNF9. GST, -Gal, and BSA were used as negative controls. Using a standard microarray scanner the nanohydrogel arrays were shown to have a consistently higher combination of absolute signal, signal-to-background ratio, and signal-to-noise ratio than any of the four microarrays. We speculate that this behavior is due to a higher density of bound protein as well as a more accessible protein conformation. Fluorescence optical microscopy can resolve individual nanohydrogels opening the possibility that assays can be scaled from arrays of 100 m diameter spots to arrays of single nanohydrogel spots. Such an advance can increase the spot density by a factor of approximately 104 and has significant implications for the highly efficient use of biological reagents in high throughput proteomic analysis.

Keywords: ELECTRON-BEAM LITHOGRAPHY; HYDROGEL; MICROARRAY; NANOARRAY; PEG; ZNF9

Document Type: Research Article

DOI: http://dx.doi.org/10.1166/jnn.2007.675

Publication date: August 1, 2007

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  • Journal for Nanoscience and Nanotechnology (JNN) is an international and multidisciplinary peer-reviewed journal with a wide-ranging coverage, consolidating research activities in all areas of nanoscience and nanotechnology into a single and unique reference source. JNN is the first cross-disciplinary journal to publish original full research articles, rapid communications of important new scientific and technological findings, timely state-of-the-art reviews with author's photo and short biography, and current research news encompassing the fundamental and applied research in all disciplines of science, engineering and medicine.
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