Nanozymes as Heterogeneous Catalysts: In Operando Aggregation and Altered Apparent Substrate Affinity Affect Catalytic Properties and Immunoassay Reliability

Nanozymes are synthetic nanoscale catalysts that are actively explored as functional replacements for natural enzymes in various applications. Often, the properties and characteristics of enzymes are directly transferred to nanozymes without considering their unique features as nanosized heterogeneous catalysts. In this article, we explore two often-neglected factors: reduced substrate binding and aggregation of peroxidase-mimicking nanozyme particles that occur in operando (i.e., during catalysis or sample incubation). A competitive Langmuir-Hinshelwood model was utilized to evaluate the impact of organic solvent on substrate binding. In operando aggregation of Au@Pt particles was confirmed by visible spectroscopy, dynamic light scattering, and ultramicroscopy. We demonstrate that both reduced substrate binding and aggregation significantly affect the kinetic parameters of nanozyme catalysis, including approximately three-fold decrease of the apparent Michaelis-Menten constant, a six-fold decrease of the maximum reaction rate, and erroneous conclusions regarding activity inhibition. The effect of aggregation was studied in microplate immunoassays and lateral flow immunoassays of Staphylococcal enterotoxin A (SEA). The results demonstrate that aggregation leads to a reduced specific signal (from immunocomplexes of SEA with Au@Pt-mAb) and an increased nonspecific signal (from aggregated Au@Pt), resulting in a lower signal-to-noise ratio. As a consequence, this leads to false-positive results in microplate immunoassays or even ruins lateral flow immunoassay performance. These results confirm that in operando colloidal stability and substrate binding are crucial factors to be considered for the accurate quantification of kinetic parameters and for various bioanalytical applications.