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.
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