In this study, the kinetic factors of the peroxidase-like MnFe2O4 nanozymes, including affinity factor, Km, and maximal velocity, Vmax, were calculated via excremental investigation. The OPD-mediated enzyme assay was utilized for determining the reaction rates and consequently constructing the saturation curves. Michaelis–Menten kinetic model was used as the standard kinetic model of enzymatic reactions while the linear plot of Lineweaver–Burk was constructed to provide reliable value for each factor. The results indicated a Vmax of 0.12 µM/sec for the peroxidase-like MnFe2O4 nanozymes. Besides, the substrate affinity factor, Km, was estimated at about 27.7 mM for the peroxidase-like MnFe2O4 nanozymes, exhibiting the high affinity of MnFe2O4 nanozymes toward OPD.

Different metals including Cr, Mn, Co, Ni, and Ag were used to synthesize the metal/ CeO2 nanoparticles. Thereafter, the peroxidase-like activity of each metal/ CeO2 nanoparticle was calculated using the TMB standard assay. Notably, the bare CoO2 nanoparticles were used as the control and its activity was considered as 100%. The results revealed that although the presence of Cr, Co, and Ag in the nanozyme structure led to enhancement of the peroxidase-like activity of the CeO2 nanoparticles, by doping the Mn and Ni in the nanozyme structure, a significant decrease in the peroxidase-like activity was observed. The Cr/CeO2 nanoparticles showed the highest activity which was 4.0-fold higher than that of the bare CeO2 and 2.0-fold higher than that of the CO/CeO2 and Ag/CeO2 nanoparticles.  

The Michaelis–Menten kinetic model was used for the evaluation of the kinetic of the nanozyme-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine over oxidase-like Ce/Fe-bimetallic nanozymes. To provide the quantitative and accurate values of kinetics parameters, Km and Vmax, the linear plot of Lineweaver–Burk was constructed. The results exhibited a Vmax as high as 67.56 nM min-1 for the oxidase-like Ce/Fe-bimetallic nanozymes. Besides, Km was found to be as low as 0.06 mM for the as-prepared nanozymes, revealing the high affinity of the nanozymes toward 3,3′,5,5′-tetramethylbenzidine. Moreover, the ratio Vmax/Km was estimated as a reliable index of catalytic efficiency of the nanozymes, revealing a high value of 1.0×10-3 min-1.

Herein, catalytic oxidation of 3,3′,5,5′-tetramethylbenzidine over NH2-MiL-88 (Fe, Ni) as the peroxidase-like nanozyme was performed, revealing high catalytic activity of the as-prepared nanozymes. The reactive oxygen species contributed in catalytic oxidation of 3,3′,5,5′-tetramethylbenzidine over NH2-MiL-88 (Fe, Ni) were identified by investigating the effect of different scavengers on the catalytic oxidation of 3,3′,5,5′-tetramethylbenzidine. The results revealed that oxygen vacancies (Ovs) and hydroxyl radicals are significantly contributed in nanozymatic reaction while by presence of 1O2, the catalytic oxidation reaction is not affected.  Considering the results, hydrogen peroxide and 3,3′,5,5′-tetramethylbenzidine were adsorbed over the nanozymes and then the active metal center of the nanozymes affected on hydrogen peroxide to generate 0OH or O2⋅-. Afterward, the oxidation of 3,3′,5,5′-tetramethylbenzidine to its corresponding colored product was occurred by the reactive oxygen species.

Herein, peroxidase-like graphene oxide/gold nanoparticles were synthesized and characterized for their peroxidase-like activity. Considering the significant effect of pH and temperature on the activity of enzyme/nanozymes, the pH and thermal stability of the peroxidase-like graphene oxide/gold nanoparticles were evaluated by determining their relative activity as an index for their stability monitoring. The results of thermal stability studies revealed a maximal activity over a wide temperature range of 20-40 ℃, revealing high thermal stability of the as-synthesized peroxidase-like graphene oxide/gold nanoparticles. Besides, the pH stability measurements revealed a maximal activity over pH= 4.5-5.5 for the as-synthesized peroxidase-like graphene oxide-supported gold nanoparticles.