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.

Key words: Glass nanopore, Glutathione S-transferase (GST), GST-GSH binding kinetics