Oxygen-Vacancy-Enhanced Peroxidase-like Activity of Reduced Co₃O₄ Nanocomposites for the Colorimetric Detection of H₂O₂ and Glucose

Colorimetric assays have drawn increasing research interest with respect to the quantitative detection of hydrogen peroxide (H₂O₂) based on artificial enzymes because of their advantages with respect to natural enzymes, including design flexibility, low cost, and high stability. Regardless, the majority of the artificial enzymes exhibit low affinity to H₂O₂ with large Michaelis-Menten constants (K_m). This indicates that the catalytic oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to blue-colored oxTMB requires a high H₂O₂ concentration, hindering the sensitivity of the colorimetric assay. To address this problem, novel reduced Co₃O₄ nanoparticles (R-Co₃O₄) have been synthesized in this study via a step-by-step procedure using ZIF-67 as the precursor. R-Co₃O₄ exhibits a considerably enhanced peroxidase-like activity when compared with that exhibited by pristine Co₃O₄ (P-Co₃O₄). The catalytic process in the case of R-Co₃O₄ occurs in accordance with the typical Michaelis-Menten equation, and the affinity of R-Co₃O₄ to H₂O₂ is apparently higher than that of P-Co₃O₄. Furthermore, the density functional theory calculations revealed that the introduction of oxygen vacancies to R-Co₃O₄ enhances its H₂O₂ adsorption ability and facilitates the decomposition of H₂O₂ to produce ·OH radicals, resulting in improved peroxidase-like activity. A simple and convenient colorimetric assay has been established based on the excellent peroxidase-like activity of R-Co₃O₄ for detecting H₂O₂ in concentrations of 1-30 μM with a detection limit of 4.3 × 10^-7 mol/L (S/N = 3). Furthermore, the R-Co₃O₄-based colorimetric method was successfully applied to glucose detection in human serum samples, demonstrating its potential for application in complex biological systems.