Hayes M A, Kuhr W G
Department of Chemistry and Biochemistry, Arizona State University, Tempe 85287, USA.
Anal Chem. 1999 May 1;71(9):1720-7. doi: 10.1021/ac980354x.
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 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.
对于基于脱氢酶的酶修饰电极的灵敏度而言，最小化过电位并产生高法拉第电流是β-烟酰胺腺嘌呤二核苷酸（NADH）快速扫描伏安法的关键问题。尽管NADH伏安法在固体电极表面表现出高过电位和较差的伏安峰形状，但电极表面的修饰可以改善碳纤维上的电化学响应。然而，在酶共价连接后，这些改善会严重退化。本研究的重点是创造改善的电子转移特性，并在整个酶连接过程中保持这些特性。开发了一种新颖的抛光和电化学预处理方法，该方法在用于NADH的碳纤维电极上产生了降低的过电位和高法拉第电流。研究了在酶制备过程中导致伏安响应退化的因素，预处理表面的老化和共价修饰都导致了这种退化。反过来，最小化制备时间的连接过程最大限度地保留了容易的电子转移特性。这些连接过程包括改变酶的表面连接反应。减少制备时间的技术包括对现有技术进行建模，然后在可能的情况下改善动力学和传质问题。替代的共价连接方法包括直接电化学胺反应和电化学还原酰肼反应。通过荧光和电化学研究证实了这些探针的表面连接和电子转移特性的保留。
