Doctor of Philosophy (PhD)
Glycoproteins have long been identified to have a profound association with human pathological processes, and they are much sought after as potential biomarkers to aid in the early diagnosis and clinical prognosis of cancers and diseases. There is currently high demand for high-throughput and low–limit–of–detection techniques that can afford profiling of the glycoproteome. Micro-total analysis systems (µTAS) based on microfluidics have the potential to fulfill these requirements, but in order to reduce the complexity of the protein pool, the µTAS devices must contain a pre-isolation and enrichment component. The research project undertaken here involved derivatization of microfluidic surfaces with ligands to allow for capture and isolation of glycoproteins in solution. It is envisioned that a microfluidic device operating in a serial affinity mode can be fabricated whereby a large set of glycoproteins are captured by a global capture element, followed by further fractionation of the previously captured glycoprotein pool into unique glycoproteins by capture elements specific to each unique protein. To that end, the research here involved (1) modification of poly(methyl methacrylate) surfaces with a boronic acid derivative as the global glycoprotein receptor and (2) investigation of a surface-amenable synthetic route for the creation of a thermoresponsive scaffold with immobilized lectin, as the specific glycoprotein receptor, and its complementary eluting sugar. Creation of these surfaces is the first step toward realizing a µTAS for glycoprotein analysis. The novel boronic acid derivative 4-[(2-aminoethyl)carbamoyl]phenylboronic acid was immobilized on carboxymethyl dextran surfaces, and its protein interaction analysis was investigated by surface plasmon resonance spectroscopy. Poly(methyl methacrylate) microfluidic surfaces were then functionalized with the novel boronic acid derivative to yield a first-generation global capture modality. Glycoprotein binding to and elution from the global capture surface was afforded using glycine- and Tris-binding buffer systems and borate-eluting buffer systems, respectively, with the aid of Tween 20. A thermoresponsive terpolymer poly(N-isopropylacrylamide–lactose–RCA120), with the lectin Ricinus communis agglutinin (RCA120) as the specific capture element, was successfully prepared by surface-amenable synthetic protocols. The synthetic strategy proposed in this work can be easily adapted in the creation of microfluidic devices that can afford the capture of specific glycoproteins.
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De Guzman, Jennifer Macalindong, "Fabrication of Receptor-Modified Microfluidic Surfaces for Applications in Glycoprotein Screening" (2010). LSU Doctoral Dissertations. 3084.
McCarley, Robin L.