Determination of the Maximum Velocity of a Peroxidase-like Nanozyme

Nanozymes are functional nanomaterials with enzyme-like activities, which have good stability and specific nanoscale properties. Among them, peroxidase-like (POD-like) nanozymes with two substrates are the biggest chunk and have been widely applied in biomedical and environmental fields. Maximum velocity (Vmax) is an essential kinetic parameter, accurate measurements of which can help in activity comparisons, mechanism studies, and nanozyme improvements. At present, the standardized assay determines the catalytic kinetics of POD-like nanozymes by a single fitting based on the Michaelis–Menten equation. However, the true Vmax cannot be confirmed by this method due to the test condition that the concentration of a fixed substrate is finite. Here, a double fitting method to determine the intrinsic Vmax of POD-like nanozymes is presented, which breaks through the limited concentration of the fixed substrate by an additional Michaelis–Menten fitting. Furthermore, a comparison of the Vmax among five typical POD-like nanozymes validates the accuracy and feasibility of our method. This work provides a credible method to determine the true Vmax of POD-like nanozymes, helping in activity comparisons and facilitating studies on the mechanism and development of POD-like nanozymes.