Hydrogel-based protein nanoarrays

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 amineterminated 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 50 nm 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 alpha-GST antibody to mediate attachment of GST-tagged ZNF9. In both cases 100 microm 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, beta-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 microm diameter spots to arrays of single nanohydrogel spots. Such an advance can increase the spot density by a factor of approximately 10(4) and has significant implications for the highly efficient use of biological reagents in high throughput proteomic analysis.