Determination of polyphenolic content in beverages using laccase, gold nanoparticles and long wavelength fluorimetry

An enzymatic fluorimetric method for the determination of polyphenol compounds in beverages is described, which is based on the temporal inhibition caused by these compounds on the oxidation of the long wavelength fluorophor indocyanine green (λex 764 nm, λem 806 nm), in the presence of the enzyme laccase and positively charged gold nanoparticles (AuNPs). The oxidation of the dye gives rise to a fast decrease in its fluorescence, but it is delayed by the polyphenol, obtaining a time period directly proportional to its concentration, which has been used as the analytical parameter. The behaviour of several benzenediols and benzenetriols in the system and the modification of the activity of the enzyme by its interaction with AuNPs have been studied.The system has been optimized using gallic acid as a polyphenol model, but the dynamic ranges of the calibration graphs and the detection limits for several of the polyphenols assayed were obtained (μmol L−1): gallic acid (0.13–5, 0.04), catechol (0.08–5, 0.01), hydroquinone (0.05–2, 0.01), hydroxyhydroquinone (0.09–5, 0.03), pyrogallol (0.17–5, 0.04). Most of the values of the regression coefficients were 0.999 and the precision of the method, expressed as RSD% and checked at two concentration levels of each analyte, ranged between 1.8 and 5.6%. The method has been applied to the determination of polyphenol content in several foodstuff samples and the results compared with those obtained with the standard Folin–Ciocalteu method.

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► A long-wavelength fluorimetric method for the determination of polyphenols is reported.
► The method involves the use of laccase, indocyanine green and gold nanoparticles.
► The delay caused by polyphenols on the enzymatic oxidation of the fluorophor is used for analytical purposes.
► The interaction of gold nanoparticles and laccase allows the improvement of the detection limits obtained.
► The method has been satisfactorily applied to the analysis of beverages.