Kinetic Profiling of Oxidoreductase-Mimicking Nanozymes: Impact of Multiple Activities, Chemical Transformations, and Colloidal Stability

In contrast to homogeneous enzyme catalysis, nanozymes are nanosized heterogeneous catalysts that perform reactions on a rigid surface. This fundamental difference between enzymes and nanozymes is often overlooked in kinetic studies and practical applications. In this article, using 14 nanozymes of various compositions (core@shell, metal-organic frameworks, metal, and metal oxide nanoparticles), we systematically demonstrate that nontypical features of nanozymes, such as multiple catalytic activities, chemical transformations, and aggregation, need to be considered in nanozyme catalysis. Ignoring these features results in the inaccurate quantification of catalytic activity. Neglecting the multiple activities led to a six-time underestimation of Mn₂O₃ oxidation activity and mischaracterization of this material as a low-active peroxidase-mimicking nanozyme. Additionally, overlooking chemical stability during catalytic reactions led to the reporting of high peroxidase-mimicking activity for Au@Ag nanoparticles, which, in reality, exhibited no intrinsic activity and oxidized the substrate through the leakage of Ag⁺ ions. Ignoring the chemical stability of Au@Prussian Blue nanoparticles may lead to more than four times overestimation of peroxidase-mimicking activity after just 24 h of storage. Finally, disregarding the colloidal stability of nanozymes led to a five-time inaccuracy in catalytic activity. These findings underscore the necessity of optimizing procedures to account for these factors in nanozyme kinetic measurements, which will in turn ensure more reliable biosensors and the success of other practical applications.