Doctor of Philosophy (PhD)
Microsystems targeted for DNA sequencing, especially those focused on electrophoretic separations, are rapidly proving their viability to genomic research, mimicking the progress made when capillary electrophoresis developed from miniaturizing slab gel electrophoresis techniques. Being the more recent electrophoretic separation platform, the commercial availability of microchip electrophoresis devices remains relatively limited. To this extent, high-aspect ratio microstructures formed in thermo plastics have been developed using rapid fabrication methods from molding tools designed for mass replication of high-aspect ratio microfeatures. In this work, the choice and compatibility of poly(methylmethacrylate) (PMMA) – the primary substrate for DNA separations in this work – was investigated for use with our fluorescence lifetime detection instrument. The accuracy and precision of the fluorescence lifetime values of dye-labeled primers used for construction of single-stranded DNA (ssDNA) sequencing tracts was determined to discern the influence of PMMA as a substrate material to the discrimination method. The separation performance of ssDNA was evaluated for potential use of the polymer-based microchip electrophoresis devices as a platform for rapid, high-throughput DNA sequencing. To enhance these separations, a scheme to modify the surface of PMMA employing chemical and photochemical methods was developed. Once optimized, a linear polyacrylamide-modified PMMA surface demonstrated an electroosmotic flow, which varied from chip to chip, lowered by two orders of magnitude and demonstrated increased efficiencies for the separation of ssDNA fragments. As part of a modular system for the analysis of DNA material being developed in our labs, a purification device fabricated in polycarbonate was used to reversibly immobilize DNA sequencing fragments. The purified ssDNA was collected and analyzed by capillary electrophoresis to evaluate the device’s efficiency in removal of contaminants from fragments constructed with dye-labeled primers. One significant result showed the necessity for a down-stream concentration method. Thus, we have investigated the use of a thermally responsive polymer, poly-N-isopropylacrylamide (pNIPAAm) grafted onto the surface of PMMA to serve as a concentration medium for the purified fragments. Results suggest pNIPAAm will be effective in concentrating and releasing fragments when changing the temperature from above its critical temperature (32°), where it exhibits a hydrophobic nature, to below it where it becomes hydrophilic.
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Llopis, Shawn, "Application of polymeric microfluidic devices for separation of single-stranded DNA" (2006). LSU Doctoral Dissertations. 2064.