The goal is to investigate a new class of materials that has to potential to provide a significant improvement in protein electrophoresis, compared to polymer gels. Today's gels give distorted, tailing zones of irreproducible position due to inhomogeneities in the gel, and they hamper the separation of hydrophobic proteins by their poor compatibility with organic solvents. The proposed work is to investigate materials comprised by ordered arrays of silica nanospheres having nanometer-scale polymer chains tethered to their surfaces. The interstitial volume serves as a sieving medium, and the polymer chains resist protein adsorption. The structural definition imposed by the silica nanospheres promises high separation efficiency and reproducibility in protein electrophoresis. The proposed work entails three parts. First, the controlled deposition of crystalline films of silica nanospheres onto plates and into channels of microfluidic chips is to be investigated. The chemical compositions of polymer coating and the nanosphere will be varied to learn how to maximize separation efficiency for proteins and base stability for the polymer and substrate. Second, the sieving transport of proteins through these mesoporous media will be studied. The migration rates and zone profiles for molecular weight standards and for fluorescence-labeled proteins will be characterized to develop a model for transport through the ordered medium. The adsorptive properties of the material will be probed by fluorescence correlation spectroscopy to determine the mobile and immobile fractions of proteins, and the rate constants for any desorption processes. Third, isoelectric focusing will be investigated to understand the roles of polymer functional group, carrier solvent, and position of the immobiline, in promoting protein solubility. Fluorescence correlation spectroscopy will probe mobile and immobile fraction. Two-dimensional electrophoretic separations will be investigated, primarily sieving and isoelectric focusing for comparison with gels, and also sieving and pore-size gradients, and electrochromatography with electrophoresis. The proposed work addresses the central issues in protein transport through crystalline mesoporous media to lay the groundwork for development of powerful new protein separations.
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