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.
In this work, chicken egg white protein
(CEW)-protected gold nanoclusters
(CEW-AuNCs) were prepared from CEW and HAuCl
4
to measure
the Cu(II)-induced prooxidant activity of antioxidant compounds such
as epicatechin, epigallocatechin gallate, catechin, rosmarinic acid,
resveratrol, ascorbic acid, and glutathione. These compounds reduced
Cu(II) to Cu(I), and the latter was mainly bound to thiol groups in
the CEW-AuNC structure. As the protein-bound Cu(I) may act as a catalytic
center for generating reactive oxygen species, the Cu(II) reducing
ability of antioxidants is an indirect measure of their prooxidant
potency. The bound Cu(I) may be released with the cuprous-selective
ligand neocuproine (Nc), forming the basis of a spectrophotometric
method measuring absorbance at 450 nm wavelength of the Cu(I)–Nc
chelate. The developed method involved a one-pot synthesis and determination
without preseparation and was applied to binary synthetic mixtures
of studied antioxidant compounds and to certain herbal plant (green
tea, linden, echinacea, and artichoke leaf) extracts to determine
the total prooxidant activities. The obtained results were statistically
compared with those of the literature Cu(II)–Nc assay using
a calcium proteinate-based solid biosensor. The developed biosensor
was durable, reliable, easily applicable, and of low cost and wide
linear range and could determine the prooxidant activities of natural
antioxidant samples with high reproducibility.
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