Dynamics of Single-Enzyme Activity in a Nanopore Confinement

Abstract

Enzymatic reactions play a crucial role in controlling and performing a number of biological processes. The ability to directly probe catalysis of a single enzyme can potentially enable a deeper understanding of the enzymatic mechanisms that are not distinguishable in ensemble measurements. Herein we report the direct observation of the electrocatalytic process of a single enzyme molecule in nanopore confinement. Horseradish peroxidase (HRP), a heme-containing enzyme that is widely used in immunoassays and bioassay, was chosen as a model enzyme in our studies. We used nanopore-based techniques to electrochemically capture a single HRP molecule in its confined space, which enable long-term observation of only a single enzyme molecule. Then the bipolar electrochemical reactions catalyzed by HRP enzyme were occurring at the two poles of the polarized gold layer. The enzyme-catalyzed electrochemical reactions resulting in easily distinguished ionic current signals enable us to monitor the catalytic process of single enzyme molecules. Using this system, we investigated the single HRP enzyme catalysis in the confined spaces of nanopore and found the heterogeneous catalytic activity between individual enzymes. This methodology provides a new tool to investigate the single-molecule catalysis of redox enzymes, which will lead to a better understanding of the enzymatic mechanisms and biocatalytic processes.