Determination of the Maximum Velocity of a Peroxidase-like Nanozyme

Abstract

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 (V max) 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 V max 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 V max 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 V max among five typical POD-like nanozymes validates the accuracy and feasibility of our method. This work provides a credible method to determine the true V max of POD-like nanozymes, helping in activity comparisons and facilitating studies on the mechanism and development of POD-like nanozymes.

Natural Enzyme-Inspired Design of the Single-Atom Cu Nanozyme as Dual-Enzyme Mimics for Distinguishing Total Antioxidant Capacity and the Ascorbic Acid Level

“…With the purpose of studying the kinetics of POD-like activity of Cu-N/C, the double-fitting method 46 was used to evaluate the V max value and the K m value of the POD-like activity (Figures S15 and S16), which break through the limitation of substrate concentration through additional Michaelis−Menten fitting. Compared with other previously reported carbon-based or Cu-based nanozymes, Cu-N/C had a larger V max and a smaller K m for H 2 O 2 , proving its better affinity and a faster reaction rate for H 2 O 2 (Table S3).…”

Sonochemical synthesis of graphene oxide-Ag2O nanozyme as an oxidize-like mimic for the highly sensitive detection of lithium in blood serum

“…(8) ) for a given TMB concentration range. where V 0 is the initial velocity (initial rate), [S] is the concentration of the substrate (TMB), K m is the Michaelis–Menten constant and V m is the maximal reaction velocity [103] , [104] .

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Self-Assembled Integrated Nanozyme Cascade Biosensor with Dual Catalytic Activity for Portable Urease Analysis

“…Nanozymes are a potential alternative to natural enzymes . They not only have the catalytic properties of proteases, but also have the features of easy storage, high temperature resistance, easy modification and low cost, which natural enzymes do not have. − It has a broad field of application in medical diagnosis, clinical treatment, bioanalysis, environmental monitoring, and agricultural production. , Currently, a variety of nanozymes inspired by natural enzymes have been developed, mainly divided into two categories: oxidases and hydrolases. − Among these, laccase, a type of oxidase with copper as the catalytic center, has become a popular research topic due to its environmentally friendly properties. − Phosphate hydrolases that cleave the P–O bond are typically members of the hydrolase family and related research is still in the initial stages of development. , However, most developed nanozymes typically have a single catalytic activity and rarely enable enzyme cascades. Conventional multistep operations lead to longer processing times, higher errors, secondary contamination, and material wastage, which are overcome by the integrated cascade reaction.…”