Probing Acetylcholinesterase Activity and Enzyme Inhibitor Evaluation by Electrostatic-Gating Nanopores

Abstract Acetylcholinesterase (AChE) plays a crucial role in neuron function and is closely associated with neurodegenerative diseases. Probing AChE-mediated catalytic reactions in biomimetic scenarios (e.g., micro- and nanospace) is of fundamental significance. Here, we present an artificial nanopore to investigate AChE activity by catalyzing acetylcholine (ACh) hydrolysis. In this assay, a predesigned glass nanopore facilitated the electrostatic assembly of the substrate, ACh. Under optimized conditions, AChE exhibited a high catalytic efficiency of up to 94% in a confined environment. The surface charge alteration due to the catalytic reaction was able to regulate ion-gating behavior and ionic current rectification (ICR). In addition, the Michaelis–Menten constant (Km) was calculated to be 26.45 mM, indicating the enhanced catalytic efficiency in the confinement, as compared to that of a conventional macroanalytical system (with a Km of 96.52 mM). The ionic current changes induced by the three enzyme inhibitors (donepezil, huperzine A, and berberine) suggested that donepezil exhibited the most potent inhibitory activity to AChE. Analysis of SH-SY5Y and PC12 cells treated with varying concentrations of the three inhibitors further corroborated their inhibitory capacity. Intriguingly, such biomimetic enzyme kinetic profiling and enzyme–drug interaction assays are currently inaccessible with conventional macro-analysis platforms. The nanopore sensor was successfully used for the detection of AChE activity in blood samples from patients with Alzheimer’s disease. This work should open new avenues for confined enzymatic activity assays and enzyme inhibitor screening.