Assessing acetylcholinesterase activity at a polarizable liquid|liquid interface

Electrochemical monitoring of acetylcholinesterase (AChE) activity was achieved at a polarizable liquid|liquid interface (ITIES) using ion transfer voltammetry. The approach enables direct detection of the ionic substrate acetylcholine (ACh⁺) and the hydrolysis product choline (Ch⁺) in real time, without the need for redox mediators or enzymatic cascades. This approach differs fundamentally from previous ITIES studies involving proteins, which primarily focused on protein adsorption, conformational changes, or protein-assisted ion transfer. Measurements in Trizma buffer and synthetic seawater revealed that AChE retains its catalytic activity in complex marine matrices. The inhibition of AChE by malathion was also resolved electrochemically, with concentration-dependent decreases in enzymatic activity observed. These results establish the ITIES as a versatile platform for studying enzymatic kinetics and inhibition phenomena and suggest its potential for the development of biosensors capable of detecting neurotoxic pollutants in environmentally relevant conditions.