Commonly 3,3'-diaminobenzidine and 3,3',5,5'-thetramethylbenzidine, abbirvated as DAB and TMB, in turn, have been employed as the standard nanozyme substrates for probing the nanozme-catalyzed oxidation, and the corresponding oxidation products of these substrates have been probed as the analytical identifiers for sensing/biosensing application in lab/medial scales. However, it is well-known that the affinity of these substrates toward binding to enzymes or especially, here, nanozymes is not the same, resulting in different enzyme-like activity and different kinetics indexes including Km and Vmax. Since, the protein-assisted/protected gold nanozymes (BSA-gold nanozymes) are type high powerful artificial peroxidase enzymes, Therefore, the kinetics indexes of these nanozymes including both Km and Vmax should be changed by varying their substrate. To prove this hypotheis, in this work, a comparative study was performed on the kinetics performances of BSA-gold nanozymes for enzyme-mediated oxidations of TMB and DAB. The results showed that the Km value of the as-mentioend nanozymes was 0.03 mM and 0.72 mM toward TMB and DAB, in order, revealing that the affinity of TMB for binding to the nanozyme active nodes is significantly higher than its alternative substrate, DAB due to its lower Km value. In contrast, the Vmax of the enzymatic reaction was found to be 263 nM sec-1 and 185 nM sec-1 for the nanozmye-mediated oxidation of TMB and DAB, respectively. The higher Vmax of the nanozyme-mediated oxidation of TMB revealed that the catalytic efficiency of the as-mentioend nanozymes toward TMB oxidation is characterstically higher (about 1.5-fold) than that of the DAB oxidation. The difference between the kinetic indexes of TMB and DAB may be related to their different oxidation pathways and their different reactivity. In fact, the DAB oxidizes via an n-electron irreversible oxidation pathway to produce an indamine polymer. While TMB nanozyme-mediated oxidation has occurred upon a 2-electron reversible mechanism for the production of a cation radical. These different pathways resulted in different kinetic performances.
Published Date: 2023-07-24; Received Date: 2023-06-26
