Development and evaluation of measurement and data-processing approaches for pseudoequilibrium methods involving enzyme-based biosensors.

摘要

The primary focus of this thesis is on the development and evaluation of pseudo-equilibrium methods for enzyme-based biosensors that offer reduced experimental variable dependencies and/or extended linear range. The proposed methods are designed to obtain the response corresponding to reaction of all the substrate in a small fixed volume of solution. The study was done in two steps. The first step involved the development of pseudo-equilibrium methods using a thin-layer cell. The approach was evaluated for an amperometric enzyme-based biosensor for glucose. Results show constant sensitivity for glucose concentrations up to five-fold the Michaelis constant and order-of-magnitude improvements in ruggedness to changes in selected experimental variables such as pH, temperature and sodium chloride concentration than steady-state options. Analogous results were attained for the effect of flow rate for an amperometric enzyme-based biosensor for hydrogen peroxide. The second phase of the study focuses on the development of pseudo-equilibrium methods using a rotating disk electrode. The biosensor is used in a rotating-disk mode such that data for current vs. time can be monitored with and without rotation of the sensor. Current is monitored until an initial steady-state response is obtained after which the rotation is stopped and current is continuously monitored until a second steady-state response is obtained. Integration of current vs. time data from the point rotation was stopped to the establishment of the second steady state yields the charge corresponding to reaction of substrate in the fixed volume established by the diffusion process. Evaluation of this pseudo-equilibrium option using a peroxidase sensor for hydrogen peroxide yielded a linear range extending to at least four times the Michaelis constant and a pH dependence approximately 40-fold less than steady-state currents obtained from the same data sets.