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Silica colloidal crystals as a gel media replacement
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Dissertation
Silica colloidal crystals as a gel media replacement
2013
Overview
Rigid pore networks formed using nonporous silica particles were used as an alternative separation medium for gel based electrophoretic techniques to enable high-throughput protein separations by reducing separation lengths. The miniaturization of isoelectric focusing to a length as short as 5 mm, with higher resolution than a 100-mm IPG strip, is shown for microchannels packed with chemically modified silica particles. Isoelectric focusing of prostate specific antigen revealed more charge variants in a 10-mm channel than a commercial gel strip with a 100-mm long immobilized pH gradient, with each having the same electric field of 800 V·cm-1 and pH gradient of 3–10. Shortening the channel to 5 mm gave significant drift and compression of the pH gradient. Despite this, comparable resolution for the 5- and 10-mm channels was obtainable in under 10 minutes for the charge variants of prostate specific antigen when a shallower pH gradient of 6 – 8 and a higher field of 1600 V·cm-1 were used for the 5 mm channel length. Size based separations of proteins in silica colloidal crystals in separation lengths of less than 5 mm is characterized for particle diameters of nominally 350 and 500 nm. A model is developed that relates the reduced electrophoretic mobility to the experimentally measurable porosity. The model fits the data with no adjustable parameters for the case of silica colloidal crystals packed in capillaries, for which independent measurements of the pore radii were made from flow data. Band broadening rapidly increases as the pore radius approaches the protein radius, indicating that the main contribution to broadening is the spatial heterogeneity of the pore radius. These combined results support the notion that rigid pore networks formed using nonporous silica can be used as an alternative medium for gel based techniques, and facilitates the design of new separations that would benefit from miniaturization.
Publisher
ProQuest Dissertations & Theses
Subject
ISBN
9781303605550, 1303605554
