SCANNING ELECTROCHEMICAL MICROSCOPY .17. STUDIES OF ENZYME MEDIATOR KINETICS FOR MEMBRANE-IMMOBILIZED AND SURFACE-IMMOBILIZED GLUCOSE-OXIDASE

The scanning electrochemical microscope (SECM) can be used to detect electron-transfer reactions of tip-generated species that occur at nonconductive surfaces containing a redox-active enzyme. Experiments were carried out with glucose oxidase immobilized on several substrates and the apparent kinetics of the enzyme catalysis were measured for several mediator oxidants under conditions of high D-glucose concentration. Theory for the SECM feedback current was developed to model the limiting zero- and first-order electron-transfer kinetics expected for such surface catalysis. Working curves relating the SECM feedback current to surface rate constants are presented. For substrates with glucose oxidase covalently attached to the surface or trapped within a porous membrane, the enzyme reaction was readily detected and the SECM feedback currents measured al low mediator concentration (ca. 50-mu-M) were found to exclusively fit the model for zero-order heterogeneous kinetics. However, at low enzyme surface concentration or with extensive chemical cross-linking of immobilized glucose oxidase, it was very difficult to use the SECM to detect and quantity the enzyme reaction. General guidelines for studying enzyme surface reactions with the SECM are given, and the prospects for detecting and kinetically assaying enzymes of cellular, and even subcellular, samples are discussed.