Sensing of acetylcholine by a tricomponent-enzyme layered electrode using faradaic impedance spectroscopy, cyclic voltammetry, and microgravimetric quartz crystal microbalance transduction methods

A three-enzyme layered assembly on Au electrodes or Au-quartz crystals, con sisting of horseradish peroxidase, HRP, choline oxidase, ChO, and acetylcho line esterase, AChE, is used to sense acetylcholine by the HRP-mediated oxi dation of 3,3',5,5'-tetramethylbenzidine, TMB (1), by H2O2, and the formati on of the insoluble product (2) on the respective transducers. The analyte- substrate, acetylcholine, is hydrolyzed by AChE to choline that is oxidized by ChO and O-2 to yield the respective betaine and H2O2. The amounts of ge nerated H2O2 and the resulting insoluble product on the transducers correla te with the concentration of acetylcholine in the samples. The formation of the insoluble product (2) on electrode supports is followed by faradaic im pedance spectroscopy that probes the increased interfacial electron-transfe r resistance upon the formation of 2, and by cyclic voltammetry that reflec ts electron-transfer barriers upon the formation of the precipitate. The fr equency of the Au-quartz crystal decreases as a result of the accumulation of the insoluble precipitate. The amount of insoluble product formed on the transducers is controlled by the concentration of acetylcholine and by the time interval of biocatalyzed precipitation. The generation of the insolub le product provides a means to amplify the sensing processes. Acetylcholine concentrations corresponding to 1 x 10(-5) M are easily sensed by the diff erent transducers.