Enzyme-Mediator Kinetics for Membrane= and Surface- Immobilized Glucose Oxidase

The scanning electrochemical microscope (SECM) can be used to detect electron-transfer reactions of tlp-generated species that occur at nonconductlve surfaces contalnlng a redox-actlve enzyme. Experlments were carrkd out wlth glucose oxldase lmmoblilzed on several substrates and the apparent kinetics of the enzyme catalyds were measured for several medlator oxldants under condltlons of hlgh pgiucose concentration. Theory for the SECM feedback current was developed to model the llmltlng zeroand flrst-order eiectrontransfer klnetlcr expected for such surface catalyds. Working curves relatlng the SECM feedback current to surface rate constants are presented. For Substrates wlth glucose OXC dam covalently attached to the surface or trapped wlthln a porous membrane, the enzyme reactlon was readlly detected and the SECM feedback currents measured at low mediator concentration (ca. 50 pM) were found to excluslvely flt the model for zeroorder heterogeneous kinetlcs. However, at low enzyme surface concentratlon or wlth extendve chemlcal crorrJinklng of immoblllzed glucose oxidase, It was very difficult to use the SECM to detect and quanttfy the enzyme reactlon. General guideilnes for studylng enzyme surface reactions with the SECM are given, and the prospects for detecting and klneticaily assaying enzymes of cellular, and even subcellular, samples are discussed.