Reusable alcohol oxidase-nPtCu/alginate beads for highly sensitive ethanol assay in beverages

Nanozymes (NZs) are nanoparticles that mimic the catalytic properties of natural enzymes. The present work aimed to obtain effective peroxidase mimetics (PO-like NZs), to characterize their morphological properties, estimate the kinetic parameters of NZs and evaluate the prospects of their application in analysis of ethanol. Herein, we have proposed a convenient spectrophotometric method for ethanol assay using reusable alginate beads enriched with alcohol oxidase (AO) and nanoparticles of PtCu (nPtCu) as PO-like NZs, and 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogen. The linear range for the proposed nPtCu-AO/alginate beads/TMB-based method is from 0.01 mM to 0.15 mM with a limit of detection of 3.3 μM ethanol. The method is used for the quantitative determination of ethanol in alcoholic beverages. The obtained results proved to be in a good correlation with the enzymatic reference method. These results highlight the potential of the nPtCu with PO-like activity in bioanalytical applications. The proposed method, being sensitive, economical and suitable for routine and micro-volume formats, can be used in clinical diagnostics for the detection of ethanol.