Degree

Doctor of Philosophy (PhD)

Department

Chemistry

Document Type

Dissertation

Abstract

Enzyme-responsive probes, whose fluorescence signature is selectively and uniquely changed upon activation by target enzyme analyte, are indispensable tools to illuminate, understand, and measure biological processes at a molecular level. This type of fluorescent probes has a multitude of applications, ranging from inhibitor screening, in vivo imaging of enzyme activities to cancer diagnostics, and intraoperative fluorescence imaging. Although it is possible for extant small-molecule enzyme-responsive probes to achieve high signal-to-background ratio (SBR), probes with ratiometric measurements are desperately needed, as a result of its ability to correct for any artifacts such as uneven probe localization. In this instance, the development of advanced ratiometric probe molecules allows for the expansion of our current probe toolbox for preclinical and clinical applications.

Delineated in this thesis are our advances in the construction of ratiometric probes for the quantification of human NAD(P)H:quinone oxidoreductase 1 (hNQO1) activity levels. Upregulation of this enzyme in solid tumors and its catalytic ability to accurately convert quinones to hydroquinones by two-electron reduction processes have the potential to yield a path for differentiating cells containing various hNQO1 activity levels. Success will occur by attaining three specific aims: (1) molecular mechanism-based design and multistep synthesis of probes and corresponding reporters; (2) spectral characterization of the probe/reporter system (quantum yields, selectivity, stability, Michaelis-Menten kinetic parameters, ratiometric response for quantification); and (3) utilization of fluorophores for hNQO1 quantification in cell monolayers and 3-dimensional multicellular spheroids (widefield imaging, confocal microscopy).

Date

7-7-2020

Committee Chair

McCarley, Robin L.

Available for download on Saturday, June 26, 2027

Share

COinS