Acetylcholinesterase (AChE) substrate inhibition is a critical self‐regulatory mechanism, yet its conformational dynamics at the single‐molecule level remain elusive. Here, we report a label‐free single‐molecule assay using nanopipettes that resolves AChE conformational states in real time during substrate inhibition. By monitoring the ionic current signatures across a substrate concentration range of 1–50 mM, we distinguish between single‐ and dual‐substrate binding states of individual AChE molecules. The blockade amplitude exhibits a clear nonmonotonic trend: it increases at low substrate concentrations (1–10 mM) and then decreases at 50 mM. This amplitude pattern correlates with the shift from monosubstrate to bisubstrate binding, reflecting a conformational transition from an expanded to a compact state that is consistent with the established dual‐site inhibition model. These results provide direct single‐molecule evidence linking specific substrate‐occupancy states to distinct conformations underlying AChE inhibition. This nanopipette platform offers a sensitive, label‐free strategy for probing conformational landscapes during enzyme catalysis and regulation.
Chen et al. (Sun,) studied this question.