Real-time Monitoring of Voltage-dependent Dissociation Kinetics of Glutathione S-Transferase with Functionalized Glass Nanopore

Abstract

The dynamic interaction between glutathione (GSH) and glutathione S-transferase (GST) is fundamental to cellular detoxification and redox regulation. Probing this transient binding at the single-molecule level under physiological conditions remains a significant challenge. Here, we present a label-free approach that exploits the easy modifiability of glass nanopores to create a sensing interface for real-time monitoring of GST-GSH association and dissociation. By analyzing the dwell times of individual binding events, we directly extracted the dissociation rate constant (k_off) online and systematically investigated its voltage dependence. The observed increase in k_off with applied voltage is rationalized by an electrophoretic force-induced lowering of the dissociation energy barrier, consistent with standard nanopore force spectroscopy models. This work establishes a single-molecule platform for the online, real-time study of interfacial biomolecular interactions, providing kinetic insights into the GST-GSH system that complements traditional ensemble methods.

Keywords

Glass nanopore / Glutathione S-transferase (GST) / GST-GSH binding kinetics

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