Amperometric biosensor based on laccase immobilized onto a nanostructured screen-printed electrode for determination of polyphenols in propolis

This work describes the preparation of an electrochemical biosensor for polyphenols determination in propolis samples. The biosensing scheme is based on a nanocomposite film of laccase enzyme (Lac) immobilized on gold nanoparticles (AuNPs) electrodeposited in a screen-printed carbon electrode (SPCE) modified with polypyrrole (Ppy) through an in-situ electropolymerization. The electrodeposition of the AuNPs increases the available area for Lac immobilization. The nanocomposite film (Ppy/Lac/AuNPs/SPCE) was characterized by scanning electron microscopy, energy dispersive X-ray spectroscopy and cyclic voltammetry. Polyphenols were detected in ethanolic extracts of propolis (EEP), where in presence of the Lac oxidized to the polyphenols, and so they can be reduced on the Ppy/Lac/AuNPs/SPCE by amperometry at −450 mV vs Ag/AgCl. The calibration plot showed a linear response in the concentration range from 1 to 250 μM expressed as caffeic acid, with a limit of detection of 0.83 μM. The time required for analysis was 15 min, compared to the time (85 min) by spectrophotometric methods, especially the so-called Folin-Ciocalteu method. The method exhibited good selectivity, stability and reproducibility for detecting polyphenols in propolis samples.