Optical and electrochemical characterization of biomacromolecular interactions at fluid-like interfaces.

摘要

This thesis comprises three different studies, each aimed at understanding biomacromolecular interactions at fluid-like interfaces. The first study is on sequential and competitive adsorption and interactions of nisin and selected proteins at a model oil-water interface, using total internal reflection fluorescence microscopy (TIRFM). Experimental results showed strong evidence of significantly enhanced adsorption of some proteins in the presence of nisin. Apparent interfacial concentrations of beta-casein were three times higher in the presence of nisin than when the pure protein adsorbed by itself under identical conditions. Enhanced adsorption was also observed for lysozyme and bovine serum albumin (BSA) in the presence of nisin, compared to adsorption of each protein by itself. Very little enhanced adsorption was observed for fibronectin in the presence of nisin. The exact mechanism for adsorption enhancement by nisin is not clear at this time. However, the experimental evidence suggests strongly that molecular weight, size of the adsorbing protein, and bulk protein concentration are all important factors.; The second study concerned the development of a flow cell to enable simultaneous optical and electrochemical characterization of biomimetic interfaces. Modifications were made to our existing optical flow cell to incorporate the capability to conduct cyclic voltammetry (CV) measurements using a three-electrode system. The feasibility of the simultaneous optical-electrochemical set-up was tested using the dye resorufin. On potential scans towards increasingly negative voltages, the fluorescent resorufin was reversibly reduced to non-fluorescent dihydroresorufin, coincident with a decrease in fluorescence emission intensity. Similarly, upon scanning towards increasingly positive voltages, the dihydroresorufm was reversibly oxidized to resorufin, resulting in an increase in fluorescence emission.; The third study outlines the process involved in the development and characterization of a fructose dehydrogenase (FDH) biosensor based on indirect bioelectrocatalysis, using coenzyme Q6 as the mediator. FDH, a naturally membrane-bound enzyme, was incorporated along with Q6 into a bilayer lipid membrane (BLM) deposited on indium tin oxide (ITO). The sensor was evaluated by using CV to monitor the change in anodic current upon addition of D-fructose. (Abstract shortened by UMI.)