Fluorescent molecular probes for quantitative biomolecular detection and cellular imaging

Over the last decade, fluorescence based techniques have become the integral tool in biology and biomedical sciences. Fluorescence detection offers several advantages such as high sensitivity, reduced chemical waste compared to either chemiluminescence or chromogenic detection systems and ability to multiplex thereby simultaneously identifying several target molecules. Both organic and inorganic fluorophores have been actively researched and used for fluorescence detection. In this dissertation work, we developed new innovative molecular probes and technologies to utilize these probes for quantitative biomolecular imaging and analysis. The first part of the work focuses on construction of a molecular probe for continuous glucose monitoring (CGM) through ratiometric FRET (fluorescence resonance energy transfer) measurements. A microsensor was developed using this molecular probe. We demonstrated that the sensor performed well for CGM. The potential application of this technology is in the development of implantable sensor for continuous monitoring of glucose in human blood or body fluids. The sensor can also be potentially coupled with insulin delivery pump to form a closed-loop insulin delivery system for diabetes treatment. A modified sensor which could potentially employed to detect very low concentration of glucose in body fluids like tear and saliva was also developed here. The second part of the work is to demonstrate the utility of the molecular probe developed to visualize the glucose within living cells through FRET imaging microscopy measurement. We showed the glucose dynamics can be quantitatively determined within living cells using this probe. This study can potentially offer new data for elucidating mechanism underlying glucose homeostasis in human blood. It will also help to understand the development of insulin-resistance in obesity and type II diabetic patients. The third part of the dissertation work is the development of frequency-domain based fluorescence lifetime imaging measurement (FLIM) technique for continuous monitoring of glucose and its dynamics within living cells. The fourth part of the dissertation work is to explore the application of quantum dots for quantitative imaging and analysis in living cells. The fifth part of the dissertation work focuses on development of rapid and automated detection techniques using quantum dots.;Key words: Biosensor, CGM, Continuous glucose monitoring, Diabetes, FLIM, Fluorescence Lifetime Imaging, Frequency Domain, FRET, fluorescence ratiometric intensity imaging microscopy, FRET molecular probe, GBP, Glucose, Influenza, Intracellular glucose detection, Lateral flow assay, Quantitative imaging, Quantum dot.