Doctor of Philosophy (PhD)
Aptamers have emerged as potential affinity agents that rival or complement antibodies in developing diagnostic assays for disease detection. We have demonstrated the use of PMMA microfluidic devices for conducting rapid affinity microchip CGE of a target protein using aptamers as the affinity probes with an electrophoresis development time of <2 min. Migration times were reproducible for thrombin complexes and free aptamers in CGE buffer with device-to-device RSD variations below 10%. By removing salts from plasma and adding an unlabeled random sequence oligonucleotide to the plasma, thrombin was detected in plasma. This method can be easily adapted to high-throughput parallel screening of plasma samples in multi-channel polymeric microdevices. The expression of rEpCAM in bacteria and mammalian cells was investigated with results showing successful expression in the mammalian systems, while protein expression in the bacteria was inhibited. Higher and prolonged expression levels were obtained in Cos 7 cells as compared to BHK and Hep2 cells due to episomal expression in Cos 7 cells. By combining a one-step IMAC procedure with electrophoresis and electro elution a more selective approach to obtaining electrophoretically pure rEpCAM was achieved. We developed dual-aptamer sandwich assays for thrombin and PDGF-BB targets on PMMA substrate using UV modification with a one-step EDC/NHS coupling reaction. Results indicate that the use of high EDC concentrations lead to faster immobilization and increased coverage density with a shorter sandwich assay development time. The assays were sensitive for both proteins and showed linearity in fluorescence response to changing protein concentration. Single molecule detection methods provide high sensitivity techniques in which aptamers can find use. We have demonstrated the use of two aptamers in a FRET-based assay for the detection of low levels of thrombin. Sensitivity and reliability of the aptamer smFRET assay depends on complex formation and stability. Due to the dissociation constants of the aptamers, single molecule events detected were below the calculated value for a 6 pM concentration of thrombin. Future work will involve adjusting the single molecule system by the use of pinholes to enable analysis of higher sample concentrations.
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Obubuafo, Anne, "Utilization of Aptamers as Affinity Probes in Polymeric Microdevices for Disease Management and the Production of a Recombinant Membrane Protein" (2008). LSU Doctoral Dissertations. 3805.
Soper, Steven A.