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, consisting of horseradish peroxidase, HRP, choline oxidase, ChO, and acetylcholine esterase, ACHE, is used to sense acetylcholine by the HRP- mediated oxidation of 3,3',5,5'-tetramethylbenzidine, TMB (1), by H'2O'2, and the formation 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 O₂ to yield the respective betaine and H'2O'2. The amounts of generated H'2O'2 and the resulting insoluble product on the transducers correlate with the concentration of acetylcholine in the samples. The formation of the insoluble product (2) on electrode supports is followed by faradaic impedance spectroscopy that probes the increased interfacial electron-transfer resistance upon the formation of 2, and by cyclic voltammetry that reflects electron-transfer barriers upon the formation of the precipitate. The frequency 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 insoluble product provides a means to amplify the sensing processes. Acetylcholine concentrations corresponding to 1 x 10^-5 M are easily sensed by the different transducers.