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 (koff) online and systematically investigated its voltage dependence. The observed increase in koff 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.
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