Doctor of Philosophy (PhD)
The need for rapid, portable and high-throughput systems in proteomics is now prevalent because of demands for generating new protein-based disease biomarkers. However, 2-D protein profile patterns are lending themselves as potential diagnostic tools for biomarker discovery. It is difficult to identify protein biomarkers which are low abundant in the presence of highly abundant proteins, especially in complex biological samples like serum. Protein profiles from 2-D separation of the protein content of cells or body fluids, which are unique to certain physiological or pathological states, are currently available on internet databases. In this work, we demonstrate the ability to separate a complex biological sample using low cost, disposable, polymer-based microchips suitable for a multidimensional techniques that employed sodium dodecyl sulfate micro-capillary gel electrophoresis (SDS µ-CGE) in the 1st dimension and micellar electrokinetic capillary chromatography (MEKC) or microemulsion electrokinetic capillary chromatography (MEEKC) in the 2nd dimension. The peak capacity generated by this microchip technique was about 3-fold greater compared to conventional 2-D separation methods and the complete separation time was 60X faster. To minimize electroosmotic flow effects, we dynamically coated the channels with methylhydroxyethyl cellulose. Proteins were detected by laser-induced fluorescence following their labeling with dyes. To mitigate challenges posed by labeling the proteins, we investigated the use of a label-free technique that relied upon conductivity measurements. Preliminary data are presented on the fabrication of on-chip electrodes using a conductive SU-8 polymer via lithography.
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Osiri, John K., "Platforms and Protocols for the Multidimensional Microchip Electrophoretic Analysis of Complex Proteomes" (2009). LSU Doctoral Dissertations. 1816.