Preservation of NADH voltammetry for enzyme-modified electrodes based on dehydrogenase

Minimizing overpotential and generating high faradaic currents are critical issues for fast-scan voltammetry of beta-nicotinamide adenine dinucleotide (NADH) for the sensitivity of enzyme-modified electrodes based on dehydrog enases. Although NADH voltammetry exhibits high overpotential and poor volt ammetric peak shape at solid electrode surfaces, modification of the electr ode surface can improve the electrochemical response at carbon fibers. Howe ver, these improvements are severely degraded upon the covalent attachment of enzyme. The creation of improved electron-transfer properties and the re tention of these properties throughout the enzyme attachment process is the focus of this study. A novel polishing and electrochemical pretreatment me thod was developed which generated a decreased overpotential and a high far adaic current at carbon-fiber electrodes for NADH. Factors that lead to a d egradation of voltammetric response during the enzyme fabrication were inve stigated, and both the aging and the covalent modification of the pretreate d surface contributed to this degradation. Attachment processes that minimi zed the preparation time, in turn, maximized the retention of the facile el ectron-transfer properties. These attachment processes included varying the surface attachment reactions for the enzyme. Preparation time reduction te chniques included modeling existing techniques and then improving kinetic a nd mass transport issues where possible. Alternate covalent attachment meth ods included a direct electrochemical amine reaction and an electrochemical ly reductive hydrazide reaction. The surface attachment and retention of el ectron-transfer properties of these probes were confirmed by fluorescence a nd electrochemical studies.