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 β-nicotinamide adenine dinucleotide (NADH) for the sensitivity of enzyme-modified electrodes based on dehydrogenases. Although NADH voltammetry exhibits high overpotential and poor voltammetric peak shape at solid electrode surfaces, modification of the electrode surface can improve the electrochemical response at carbon fibers. However, these improvements are severely degraded upon the covalent attachment of enzyme. The creation of improved electron-transfer properties and the retention of these properties throughout the enzyme attachment process is the focus of this study. A novel polishing and electrochemical pretreatment method was developed which generated a decreased overpotential and a high faradaic current at carbon-fiber electrodes for NADH. Factors that lead to a degradation of voltammetric response during the enzyme fabrication were investigated, and both the aging and the covalent modification of the pretreated surface contributed to this degradation. Attachment processes that minimized the preparation time, in turn, maximized the retention of the facile electron-transfer properties. These attachment processes included varying the surface attachment reactions for the enzyme. Preparation time reduction techniques included modeling existing techniques and then improving kinetic and mass transport issues where possible. Alternate covalent attachment methods included a direct electrochemical amine reaction and an electrochemically reductive hydrazide reaction. The surface attachment and retention of electron-transfer properties of these probes were confirmed by fluorescence and electrochemical studies.