A novel amperometric biosensor for rapid detection of ethanol utilizing gold nanoparticles and enzyme coupled PVC reaction cell

This research was aimed at the fabrication of an improved enzyme-based amperometric biosensor for rapid quantification of ethanol. Alcohol oxidase (AOX) from Pichia pastoris was covalently immobilized on chemically treated polyvinylchloride (PVC) beaker and subsequently horseradish peroxidase (HRP), nafion (Nf), carboxylated multi-walled carbon nanotubes (c-MWCNTs), chitosan (CHIT) and gold nanoparticles (AuNPs) were immobilized onto Au electrode to fabricate a working electrode. The enzyme-coated PVC surface was analysed morphologically via scanning electron microscopy (SEM). At different stages of construction, the electrochemical properties of working electrode were deciphered by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor displayed optimal response in a short time span of 12 s at pH 7.5 and 35°C temperature. The working range exhibited by the proposed biosensor was 0.01-42 mM with a limit of detection (LOD) of 0.0001 µM and storage stability of 180 days at 4°C. When level of alcohol was evaluated in commercial samples via standard assay kit and existing biosensor, a good correlation (R² = 0.98) was observed which authenticates its reliability.