DEVELOPMENT OF ELECTROCHEMICAL AND SPECTROELECTROCHEMICAL BIOSENSORS FOR GLUCOSE

Abstract

This thesis concerns the development of amperometric enzyme electrodes and fiber-optic spectroelectrochemical enzyme probes for glucose in the continuous-flow analysis. The amperometric studies provide a basic understanding for the development of a reversible spectroelectrochemical enzyme optrode. The performance of an enzyme electrode that monitors the oxygen depletion in the enzymatic reaction was found to be dependent on the sensitivity of the electrode material to hydrogen peroxide reduction. The choice of operating potential is also important to minimize the effects of electroactive interferences. Tetrothiafulvalene serves as a good electron mediator for glucose oxidase in its application to second-generation enzyme electrodes. Fast homogeneous electron exchange rate with glucose oxidase was obtained. The fabrication and characterization of tetrathiafulvalene-glucose oxidase enzyme electrodes in continuous-flow analysis is described. No preconditioning of the electrode is required, and the electrode fabricated from carbon paste method gives better results compared with the droplet evaporation of tetrathiafulvalene. The simplified carbon paste approach is used in preparing the tetrathiafulvalene-7,7,8,8-etracyanoquinodimethane base glucose oxidase electrodes. The electrochemical rate constant k'ME obtained is comparable to the literature value, The presence of carbon in the paste enhances the electrode stability. First-, second- and third-generation amperometric glucose enzyme electrodes were compared in the monitoring of undiluted whole blood using a wall-jet detector. Characterization of the wall-jet cell show that due consideration must be given to the flow cell geometry and orientation of the enzyme electrode with respect to the jet inlet. First-generation electrodes, based on the detection of hydrogen peroxide at platinum electrode, are generally unsuitable because of the eventual electrode poisoning and their susceptibility to oxygen variation. Second-generation electrodes are more suitable for the analysis of whole blood. However, the choice of mediator is important. A third-generation electrode based on tetrathiafulvalene tetracyanoquinodimethane proved useful, but showed lower stability and dynamic range compared to the second-generation devices. The clinical evaluation of the carbon paste-tetrathia-fulvalene enzyme electrode gave a good precision(< 2% in the flow-injection mode), and recovery (about 100%). The electrode validated in clinical analysis yields linear regression equations of Y = 1.152X + 0.295 and Y = 0.979X + 0.500 for flow-injection and steady-state modes respectively. The electrode response was found to be relatively unaffected by anticoagulants, blood preservatives and interfering susbtances. Pulsed amperometric detection of glucose using tetrathiafulvalene mediated enzyme electrode shows a higher precision in long term flow-injection analysis. The method is less susceptible to the flow-rate. However, the current was lower and the detention limits higher compared with do amperometric mode. An electrochemically generated tetrathiafulvalene redox mediator provides a convenient and reversible indicator reagent for a glucose fiber-optic sensor. The sensor was operated under continuous-flow conditions with a thin-layer spectroelectrochemical flow cell. The fiber-optic detection is less prone to interfering species and dissolved oxygen compared to amperometric detection.