Unveiling the Actual Catalytic Sites in Nanozyme‐Catalyzed Oxidation of o‐Phenylenediamine

Nanozymes have offered remarkable advantages over natural enzymes and found widespread applications including biosensors, immunoassays, nanomedicines, and environmental remediation. Oxidation of o‐phenylenediamine (OPD) by nanozymes has been listed as a standard protocol for determining nanozyme activities. Given the complexity of OPD oxidation processes, however, the mechanism of nanozyme‐catalyzed oxidation of OPD remains elusive. In this report, mechanistic studies of nanozyme‐catalyzed oxidation of OPD are performed and a distinguishably different mechanism from that of natural enzymes is found. A combination of Fourier transform infrared spectroscopy, nuclear magnetic resonance, electrospray ionization mass spectrometry, and electron microscopic studies provides compelling evidence that polymerization of OPD occurs on the surface of several different nanozymes. The unexpected polymerization causes a dense coating layer of poly(o‐phenylenediamine) (POPD) on nanozymes renders the intrinsic properties of nanozymes. Therefore, this fundamental discovery raise serious concerns using OPD‐based colorimetric method for determining nanozyme activities. Without examining the surface change of nanozymes after catalytic reactions, the use of OPD‐based colorimetric method for determining nanozyme activities is strongly discouraged. Furthermore, POPD is discovered as a new oxidase mimic, and this new mechanism also provides a general and robust method to coat nanomaterials with POPD polymers of enzyme‐mimicking properties. In the process of nanozyme‐catalyzed oxidation of o‐phenylenediamine (OPD), oxidation polymerization of OPD occurs on the surface of nanozymes. Related characterizations reveal that the formed coating layer is poly(o‐phenylenediamine) (POPD), which is confirmed to affect and render the intrinsic properties of nanozymes. Serious concerns should be taken when using OPD‐based colorimetric method for determining nanozyme activities.