To develop a protein-based biosensor measuring the pro-oxidant activities of phenolic compounds, egg white proteins were precipitated with calcium chloride to obtain an insoluble calcium proteinate complex. This biosensor was used for the determination of Cu(II)-induced pro-oxidant activity of antioxidants such as gallic acid, catechin, epicatechin, quercetin, chlorogenic acid and myricetin, and ascorbic acid. This assay involved the reduction of Cu(II) ions to Cu(I) by antioxidant compounds (simultaneously giving rise to reactive oxygen species) and binding of the formed Cu(I) to the solid biosensor. The protein-bound Cu(I), an indicator of pro-oxidant activity of antioxidants on proteins, was colorimetrically determined at 450 nm with neocuproine (Nc). The method was applied to synthetic mixtures and herbal (sage, green tea, mint, and marjoram) infusions, and its findings were compared to those of a modified carbonyl detection assay. This low-cost biosensor can be prepared in large quantities and used for a long time.
Keywords: carbonyl assay; copper detection; cupric neocuproine; phenolics; pro-oxidant biosensor; protein precipitation.