Colorimetric assay based on horseradish peroxidase/reduced graphene oxide hybrid for sensitive detection of hydrogen peroxide in beverages

Abstract

We reported a simple and sensitive colorimetric assay for detection of hydrogen peroxide (H₂O₂) based on the oxidation of 2,2׳-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) by UV-Vis spectroscopy method. The reduced graphene oxide (rGO) was prepared using green tea extract as bio-reducing and stabilizer agent and decorated by horseradish peroxidase (HRP). The surface of Au interface was modified with HRP-rGO hybrid. The formation of HRP-rGO hybrid was confirmed by cyclic voltammetry, scanning electron microscopy (SEM), energy-dispersive X-ray Spectroscopy (EDX) and Raman spectroscopy·H₂O₂ can be catalysed by HRP-rGO hybrid and converted into water and oxygen. The ABTS substrate takes up oxygen to form a green coloured product that has absorption peaks at 421, 655 nm and 737 nm. The colour development is linearly dependent on HRP in the range of 4-50 µg/L. The color of the green product solution is stable for 20 min. The absorption intensity is strongly related to the hydrogen peroxide concentration. The absorption intensity of the formed product scaled linearly with the hydrogen peroxide concentration in the ranges of 0.3-20 µM and 20-8000 µM with a detection limit of ≈15 nM could be achieved. The biosensor with excellent limit detection and wide linear ranges was adapted to monitor H₂O₂ in different beverages.

Keywords: Green tea extract; Horseradish peroxidase; Hydrogen peroxide; Reduced graphene oxide.