Experimental and In Silico Studies on the Development of an Electrochemical Biosensor for the Quantification of H2O2 Based on the ChOx Enzyme

This work presents the development of a biosensing platform for hydrogen peroxide (H2O2) electrochemical reduction. The developed platform uses a multi-walled carbon nanotube paste (PMWCNT) and the enzyme cholesterol oxidase (ChOx). The supramolecular architecture of the PMWCNT/ChOx platform was characterized using cyclic voltammetry, electrochemical impedance spectroscopy, and amperometry. The results indicated that the presence of ChOx enhances the sensitivity of electrochemical detection for H2O2 by 21 times compared to that without ChOx. The designed electrochemical sensing bio-platform for H2O2 shows a sensitivity of 26.15 µA/mM in the linear range from 0.4 to 4.0 mM, an LOD of 0.43 µM, and an LOQ of 1.31 µM. Furthermore, in silico studies (molecular dynamics simulations, molecular docking assays, and binding free energy calculations (ΔGb)) were carried out to characterize and validate the molecular interaction between ChOx and H2O2. The computed data confirmed that the binding is spontaneous, and the type of labile interaction promotes a rapid electrochemical reduction of H2O2.