A direct and simple fluorescent assay for the total polyphenol determination based on the bioconjugate formed between the laccase enzyme (TvL from Trametes versicolor) and carbon nanodots (CD) is developed. One of the most used reactions for the determination of phenols is based on the enzymatic reaction of their oxidation to quinones. In this work, CD has been biofunctionalized with TvL (TvL-CD) and employed as a fluorescent label to follow the enzymatic reaction. The bioconjugate was formed and characterized by spectroscopy and microscopy. The optimal TvL-CD ratio was established. The reaction between the bioconjugate and a phenolic compound such as gallic acid (GA) was followed by monitoring the fluorescence bioconjugate decrease due to the quenching effect of the quinones generated in the enzymatic reaction. These studies confirm that bioconjugation does not inhibit the enzymatic activity and the fluorescence decrease during the enzymatic reaction is mainly due to an electron transfer processes. Based on these results, a new method for the quantitative determination of polyphenols measured as GA concentration is developed. The detection and quantification limit was found to be 7.4 and 25 μM, respectively. Subsequently, the method has been applied to the direct determination of GA in wine, juice, and rice leaf extracts.
Keywords: Bioconjugate; Carbon nanodots; Fluorescent label; Laccase enzyme; Polyphenol determination.
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