Electrochemical Methodology for Single Molecule Enzymology

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.