Doctor of Philosophy (PhD), Ohio University, 2023, Chemistry and Biochemistry (Arts and Sciences)

In recent years, there took place a notable advancement observed in single-molecule fluorescence microscopy methods and its use in various biomolecular research. This technique allows for direct visualization of dynamics and its detailed complexities of various biological processes at the molecular level which is not possible in bulk measurement. Usually, in experiments related to single-molecule fluorescence measurements, the abundance of key molecules is intentionally minimized, which reduces the noise and improves the quality of imaging. However, such a strategy does not work when experiments involve weak interaction between biomolecules. In such a situation nonspecific interaction between molecules of interest and glass would lead to an unwanted fluorescence background signal, which compromises the imaging quality and reduces the measurement accuracy. In this work, glass surfaces have been functionalized in multiple steps. In the initial step, the glass coverslip surface is modified with (3-aminopropyl) triethoxysilane (APTES), and in the next step, the surface is functionalized using methoxy-terminated polyethylene glycol (mPEG) and biotin-terminated polyethylene glycol (bPEG) molecules. Each surface is characterized using dye-labeled protein molecules called neutravidin. for a variety of single-molecule fluorescence studies as PEG molecules are known to repel any nonspecific molecules binding on the functionalized surfaces. Then the surface is used for two-end immobilization of lambda DNA using biotin and neutravidin interaction. Once the surface is functionalized and characterized, lambda DNA is two ends immobilized on the surface using biotin and neutravidin interaction. Then we use that platform to study the intercalation and de-intercalation kinetics of various intercalating dyes such as single- intercalator (YO-PRO-1) and doubleintercalator (YOYO-1) at various experimental conditions of ionic strength and flow speed of the buffer (open full item for complete abstract)

Committee: Jixin Chen (Advisor) Subjects: Chemistry; Physical Chemistry

PhD, University of Cincinnati, 2009, Arts and Sciences : Chemistry

A rapid and sensitive fluorimetric assay has been developed to determine the biotin-binding capacities (BBC) of streptavidin or NeutrAvidin™ coated solid supports and therefore to estimate immobilized streptavidin/NeutrAvidin™ concentration. Biotin-4-fluorescein (b-4-f) on binding with streptavidin/NeutrAvidin™ gets quenched. A series of titrations were done using b-4-f as titrant for streptavidin/NeutrAvidin™ solid supports. Break points of titrations were used to determine total BBC. This method was successfully tested on four different solid supports – 2.8 µm streptavidin magnetic beads (4 × 106 biotin/bead), 2.8 µm NeutrAvidin™ magnetic beads (2 × 106 biotin/bead), 0.31 µm non-magnetic streptavidin polystyrene blue particles (6 × 103 biotin/particle) and a 96 well streptavidin micro-titer plate (5 × 1013 biotin/well). Another Fluorimetric method has been developed to determine the ligand binding capacities of streptavidin/NeutrAvidin™ coated solid supports. Streptavidin/NeutrAvidin™ coated solid supports were titrated with biotinylated antibodies followed by addition of b-4-f. Antibody concentration at the asymptote intersection (saturation point) was used to determine the ligand-binding capacity of four different solid supports as above. Streptavidin (2.8 µm) beads showed approx. 15fold decrease, streptavidin particles (0.31 µm) had 4fold decrease while streptavidin coated polystyrene plates, exhibited approx. 25fold decrease in biotinylated antibodies than their respective BBC published by manufacturers. Method can also be applied to determine the unknown concentration of biotinylated ligand in the solution. A calibration graph of ‘streptavidin high binding capacity coated plate' vs. biotinylated antibody showed the linear range as 0.07 – 1.2 µg/well (4 – 65 nM) and a detection limit of 0.5 nM. The effect of different generations of dendrimers on concentration of immobilized biotinylated ligands was studied. PAMAM dendrimer of generations G5.5 and G6.5 were (open full item for complete abstract)

Committee: H Brian Halsall PhD (Committee Chair); William Heineman PhD (Committee Chair); Patrick Limbach PhD (Committee Member); Edward Merino PhD (Committee Member) Subjects: Biochemistry