Doctor of Philosophy (PhD)
The development of fluorogenic substrates for real-time tumor cell detection has led to a vastly expanding field for personal oncology. Fluorophores have been studied as appendages to larger scaffolds leading to accumulation of these dyes in tumor cells or their surrounding environment, taking advantage of tumor anatomy. A new class of fluorophores has been developed in which the dye is an active participant in the mechanism of cancer cell detection. These dyes have been conjugated such that their fluorescence has been eliminated or altered and will undergo a change to reveal their fluorescent signal upon activation by a mechanism that is unique to tumor cells. The research presented in this dissertation encompasses the design, synthesis, properties, and utilization of latent fluorophores that are specifically activated by an enzyme that is highly upregulated in tumor cells, NAD(P)H:quinone oxidoreductase-1 (NQO1). These dyes utilize the 2-electron reduction of quinones to hydroquinones, which NQO1 specifically catalyzes. A dye’s fluorescence can be quenched by conjugating a quinone directly to the fluorophore, only to have its signal uncloaked after activation by NQO1. The objectives in this research will be achieved by: (1) the characterization of properties (stability in biological environments, quantum yields) of the quinone, dyes, and their conjugated counterparts; (2) determination of kinetic parameters (Michaelis constant (Km), theoretical maximum velocity (Vmax), catalytic constant (kcat), enzyme efficiency (kcat/Km) of the substrates towards NQO1 and the way solvent affects such parameters during assay conditions; and (3) utilization of a latent fluorophore for in vivo NQO1 analysis (widefield imaging, confocal single-/two-photon microscopy, flow cytometry) and determining the fate of the released fluorophore. Integration of these studies led to the development of two different latent fluorophores that are readily activated by NQO1. Of these two fluorogenic cancer sensors, one was found to possess a highly novel quenching mechanism between the quinone and the dye.
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Silvers, William, "Design and Analysis of Cloaked Fluorophores for Rapid Detection and Visualization of Cancer Cells Containing NAD(P)H:Quinone Oxidoreductase-1" (2012). LSU Doctoral Dissertations. 3974.