International Research Fellowship Program: Electrochemical Biosensors for the Detection of Individual Enzyme Catalysis Events

国际研究奖学金计划使美国科学家和工程师能够在国外进行9到24个月的研究。该计划的奖项提供了联合研究的机会，并利用国外独特或互补的设施、专业知识和实验条件。该奖项将支持埃德加·D·戈卢赫博士与荷兰代尔夫特大学的Serge Lemay博士合作的为期24个月的研究奖学金。该计划的目标是开发一种新技术，通过在每次产生产品分子时提供信号来实时电化学检测单一酶的活性。该方法展示了具有单分子检测灵敏度的新一代电化学生物传感器，该传感器将用于研究单个酶分子的内部动力学。这项技术使用了一种微型制造设备，该设备由玻璃通道中的两个电极组成，两个电极之间的距离约为10纳米。电极检测位于它们之间充满液体的体积中的电化学活性分子。催化是通过在电极之间放置一种单一的酶来研究的，这种酶可以将不活跃的底物转化为氧化还原活性产物。可逆氧化还原活性分子在暴露在电势下时可以迅速获得和失去电子。虽然单个电子转移事件很难检测到，但纳米级的间隙允许每秒通过氧化还原活性分子传输数百万电子，从而产生可测量的电流。这种检测方案提供的电化学信号的显著放大使得构建具有前所未有的灵敏度和选择性的电化学生物传感器成为可能。以酪氨酸酶为模型体系，催化酚类物质转化为氧化还原活性的苯二酚分子。特别令人感兴趣的是酶随着时间的推移所经历的结构变化及其对酶活性的影响，以及外部因素的影响，如pH、温度和缓冲液浓度。最后，研究了系统微小液体体积对底物浓度剧烈波动的影响。

0754396GoluchThe International Research Fellowship Program enables U.S. scientists and engineers to conduct nine to twenty-four months of research abroad. The program's awards provide opportunities for joint research, and the use of unique or complementary facilities, expertise and experimental conditions abroad.This award will support a twenty-four month research fellowship by Dr. Edgar D. Goluch to work with Dr. Serge Lemay at Delft University in the Netherlands.The goal of the proposal is to develop a new technique that electrochemically detects the activity of a single enzyme in real time by providing a signal each time a product molecule is generated. The approach demonstrates a new generation of electrochemical biosensors with single molecule detection sensitivity that will be employed to study the internal dynamics of individual enzyme molecules. This technique uses a microfabricated device that consists of two electrodes in a glass channel that are separated by about 10 nanometers. The electrodes detect electrochemically active molecules that are located in the liquid-filled volume between them. Catalysis is studied by placing a single enzyme that can convert inactive substrates into redox-active products between the electrodes. A reversibly redox-active molecule can rapidly gain and lose electrons when exposed to an electrical potential. While a single electron transfer event is very difficult to detect, the nanoscale gap allows millions of electrons to be transported by a redox-active molecule every second, thus producing a measurable amount of electrical current. The dramatic amplification of the electrochemical signals provided by this detection scheme allows the construction of electrochemical biosensors with unprecedented sensitivity and selectivity. Tyrosinase, which catalyzes the conversion of phenols into redox-active quinone molecules, is used as the model system. Of particular interest are the structural changes that the enzyme undergoes over time and their effect on enzyme activity, as well as the effects of external factors, such as pH, temperature, and buffer concentration. Finally, the influence of dramatic fluctuations in substrate concentration generated by the minute fluid volume of the system is being investigated.