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

国际研究奖学金计划：用于检测单个酶催化事件的电化学生物传感器

• 批准号: 0754396
• 负责人: Edgar Goluch
• 金额: $ 14.98万
• 依托单位: Goluch Edgar D
• 依托单位国家: 美国
• 项目类别: Fellowship Award
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0754396&HistoricalAwards=false
• 关键词: International; Research; Fellowship; Program; Electrochemical

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.

0754396Goluch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九到二十四个月的研究。 该项目的奖项为联合研究以及使用国外独特或互补的设施、专业知识和实验条件提供了机会。该奖项将支持 Edgar D. Goluch 博士与 Serge Lemay 博士在荷兰代尔夫特大学。该提案的目标是开发一种新技术，通过在每次生成产物分子时提供信号，以电化学方式实时检测单个酶的活性。 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，将用于研究单个酶分子的内部动力学。 该技术使用一种微加工装置，该装置由玻璃通道中的两个电极组成，电极间隔约 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。 通过在电极之间放置一种可以将非活性底物转化为氧化还原活性产物的酶来研究催化作用。 当暴露于电位时，可逆氧化还原活性分子可以快速获得和失去电子。 虽然单个电子转移事件很难检测到，但纳米级间隙允许氧化还原活性分子每秒传输数百万个电子，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着放大允许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。 酪氨酸酶可催化酚类转化为氧化还原活性醌分子，被用作模型系统。 特别令人感兴趣的是酶随时间发生的结构变化及其对酶活性的影响，以及外部因素（例如 pH、温度和缓冲液浓度）的影响。 最后，正在研究系统的微小流体体积产生的底物浓度剧烈波动的影响。