Metal Nanoparticle-Based Amperometric Biosensors for Ethanol Determination: Comparative Study of Alcohol Oxidase and Alcohol Dehydrogenase Bioelectrodes

Rapid and reliable determination of ethanol is important in food quality control, clinical diagnostics, and industrial monitoring. In this work, amperometric biosensors based on alcohol oxidase (AO) and alcohol dehydrogenase (ADH) were developed and compared for ethanol determination. Copper (nCu) and bimetallic copper–gold (nCuAu) nanoparticles were synthesized by chemical reduction and characterized as nanozyme-like electrocatalytic materials. The catalytic activity of the nanoparticles toward hydrogen peroxide reduction was investigated and applied for the construction of AO-based biosensors. Several enzyme immobilization approaches, including glutaraldehyde vapor crosslinking, dialysis membrane entrapment, and polymer matrices (ELO, ELO/RD1, and M1-2), were evaluated. The ELO matrix provided efficient immobilization while preserving catalytic activity and was selected for further studies. Among AO-based bioelectrodes, AO/nCu/GE exhibited higher sensitivity toward ethanol than AO/nCuAu/GE. In parallel, an ADH-based biosensor incorporating NADP+ and benzoquinone as an electron-transfer mediator was developed. The ADH/ELO/GE biosensor demonstrated the highest sensitivity (305 A·M−1·m−2), a low detection limit (0.005 mM), good reproducibility (RSD = 3.5%), and acceptable storage stability. The biosensor showed negligible interference from common electroactive compounds and was successfully validated for ethanol determination in commercial wine samples. The results demonstrate the potential of enzyme–nanozyme hybrid systems as effective analytical platforms for ethanol monitoring.