We are interested in controlling the spatial distribution of proteins on surfaces at cellular and subcellular length scales. To do this, we use a variation of e-beam lithography in a field-emission scanning electron microscope (SEM) to radiation crosslink thin films of water- soluble polymers such as poly(ethylene glycol) [PEG] and poly (carboxylic acids). We can simultaneously pattern the resulting hydrogels on silicon or glass surfaces with nanoscale and microscale feature sizes. Using hydroxy-terminated PEG 6800 we create gels with swell ratios between unity and fifteen depending on the degree of radiation crosslinking, and the swelling properties can be interpreted in terms of the Flory-Rehner formulation modified for one-dimensional swelling. While lightly-crosslinked PEG gels resist protein adsorption and cell adhesion as expected, highly crosslinked PEG gels adsorb such proteins as fibronectin and laminin and consequently become adhesive to fibroblasts, macrophages, and neurons. By spatially modulating the degree of crosslinking, we can localize these cells on surfaces and, for example, direct neurite outgrowth. If instead of using hydroxy-terminated PEG we use amine- terminated PEG, we introduce the additional flexibility of creating high-swelling PEG gels that resist nonspecific protein adsorption but to which specific proteins can be covalently bound. These can be surface patterned at submicron spacings, and we can pattern 7500 nanohydrogels in a 100 micron diameter arrays in 10 seconds. This is an areal density ~104 times greater than a modern DNA/protein chip, and the required bioreagents for chip fabrication and processing are proportionately less. We can bind fibronectin and laminin to different arrays, and we show that these proteins maintain their biospecificity after binding to the nanohydrogels with high fidelity. Looking to applications in next-generation protein-chip technology, our most recent experiments compare the performance of nanohydrogel arrays to that of standard protein microarrays using oligonucleotides which specifically bind nucleic acid-binding proteins.
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