Optimizing the standardized assays for determining the catalytic activity and kinetics of peroxidase-like nanozymes

Nanozymes are nanomaterials with enzyme-like catalytic properties. They are attractive reagents because they do not have the same limitations of natural enzymes (e.g., high cost, low stability and difficult storage). To test, optimize and compare nanozymes, it is important to establish fundamental principles and systematic standards to fully characterize their catalytic performance. Our 2018 protocol describes how to characterize the catalytic activity and kinetics of peroxidase nanozymes, the most widely used type of nanozyme. This approach was based on Michaelis–Menten enzyme kinetics and is now updated to take into account the unique physicochemical properties of nanomaterials that determine the catalytic kinetics of nanozymes. The updated procedure describes how to determine the number of active sites as well as other physicochemical properties such as surface area, shape and size. It also outlines how to calculate the hydroxyl adsorption energy from the crystal structure using the density functional theory method. The calculations now incorporate these measurements and computations to better characterize the catalytic kinetics of peroxidase nanozymes that have different shapes, sizes and compositions. This updated protocol better describes the catalytic performance of nanozymes and benefits the development of nanozyme research since further nanozyme development requires precise control of activity by engineering the electronic, geometric structure and atomic configuration of the catalytic sites of nanozymes. The characterization of the catalytic activity of peroxidase nanozymes and the evaluation of their kinetics can be performed in 4 h. The procedure is suitable for users with expertise in nano- and materials technology. Key points Nanozymes are nanoparticles designed to have catalytic properties similar to those of natural enzymes. Design and optimization of nanozyme properties require analytical methods to characterize their physical properties as well as their catalytic activity and kinetics. This is an updated protocol for measuring catalytic behavior that incorporates data from measured physical properties unique to each nanoparticle as well as density functional theory calculations into the Michaelis–Menten approach. Developing optimal nanozymes requires standardized methods for measuring their catalytic activity and reaction kinetics. This protocol integrates enzyme based Michaelis–Menten kinetics with measured physical properties and computa