Electrochemical Methodology for Single Molecule Enzymology

酶是细胞内的特殊蛋白质，可以将化学反应物保持在精确的位置，以加速生化反应。虽然它们参与化学反应，但酶并没有在反应中被耗尽。在基础水平上研究这些反应特别具有挑战性，因为它需要检测单个酶。在化学系的化学测量和成像项目的支持下，杰弗里·E。位于查佩尔山的北卡罗来纳州大学的迪克正在推进研究单一酶的电化学方法。迪克教授和他的学生们一起工作，创造了微型电极，可以表征由单个酶催化的化学反应，其体积小于单个细胞。该项目有可能提供研究细胞中化学反应的新方法，并影响超灵敏生物传感器的发展。此外，这些活动为研究生和本科生提供培训机会，他们将成为下一代的电化学家和生物技术专家。该小组还在开发用于美国各地高中的电化学仪器，将该项目的影响扩大到研究实验室之外。Dick在查佩尔山的北卡罗来纳州大学开发电化学方法来检测和定量单个酶分子。单个酶产生的电流量取决于其最大周转率。常见的酶周转率为1000 Hz的量级，这意味着单个酶产生的电流量低于1毫微微安培。这种微小的电流不能用合理的带宽来检测和量化。Dick博士和他的学生们正在开发基于电位的方法来解决这一基本限制。电位测定法是一种强大的技术，因为它需要非常小的可变偏置电流来产生电位测量，并且测量本身与电极尺寸无关。单个酶的周转率的放大是通过将单个酶分子捕获在亚阿升体积内、合成为乳液或通过纳米纤维制造来实现的。在这些小体积中，有效地是单一酶生物传感器，使用本研究项目中开发的基于电化学的新型测量方法严格评估单一酶的物理化学性质。该项目还专注于有希望特异性和快速检测COVID-19等单一病毒颗粒的方法。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Enzymes are special proteins inside cells that hold chemical reactants in precise positions to accelerate bio-chemical reactions. While they are involved in chemical reactions, enzymes are not used up in the reactions. Studying these reactions at a fundamental level is particularly challenging, because it requires detecting individual enzymes. With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Professor Jeffrey E. Dick at the University of North Carolina at Chapel Hill is advancing electrochemical methods for studying single enzymes. Working with his students, Professor Dick is creating tiny electrodes that can characterize chemical reactions catalyzed by a single enzyme in volumes smaller than a single cell. The project has the potential to offer new ways of studying chemical reactions in cells and impact the development of ultrasensitive biosensors. In addition, the activities provide training opportunities for graduate and undergraduate students who will become the next generation of electrochemists and biotechnologists. The group is also developing electrochemical instruments for use in high schools around the United States, broadening the impact of the project beyond the research laboratory.With this award, the Chemical Measurement and Imaging Program is funding Dr. Jeffrey E. Dick at the University of North Carolina at Chapel Hill to develop electrochemical methods to detect and quantify single enzyme molecules. The amount of current a single enzyme produces depends on its maximum turnover rate. Common enzyme turnover rates are on the order of 1000 Hz, implying the amount of current generated by a single enzyme is under a femtoampere. This tiny current cannot be detected and quantified with reasonable bandwidth. With his students, Dr. Dick is developing potentiometric-based methods to address this fundamental limitation. Potentiometry is a powerful technique in that it requires a very small, variable bias current to produce a potential measurement, and the measurement itself is independent of electrode size. The amplification of a single enzyme’s turnover is achieved by trapping a single enzyme molecule within a sub-attoliter volume, synthesized as an emulsion or fabricated via nanofabrication. In these small volumes, effectively a single enzyme biosensor, a single enzyme’s physicochemical properties are rigorously evaluated using novel electrochemistry-based measurement methods developed in this research project. The project also focuses on methods that hold promise to specifically and rapidly detect single virus particles such as COVID-19.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.