An electrochemical method based on the reduction of MB for determining the catalytic specific activity of peroxidase-like nanozymes immobilized

Current assays for POD-like catalytic specific activity of nanozymes are often under homogeneous dispersion in solution, however, in practical applications, nanozymes are often immobilized in various media, such as gels, necessitating new methods to characterize the catalytic activity of immobilized nanozymes. This study introduces an electrochemical method to evaluate the catalytic activity of POD-like nanozymes immobilized in thin films, using iron oxide nanozymes synthesized via co-precipitation as a model. The approach normalizes the current intensity of the methylene blue (MB) reduction peak to the electrochemically active surface area, calculating the charge transfer per unit area per unit time ( μC s −1 cm −2 ) to determine specific catalytic activity. The catalytic activities for various nanozymes, including cc-Fe 3 O 4 NPs, γ-Fe 2 O 3 NPs, α-Fe 2 O 3 NPs, H-Fe 3 O 4 NPs, Fe 3 O 4 -TEG NPs, Co 3 O 4 NPs, and Prussian blue nanoparticles (PB NPs), are measured as 38.18, 7.78, 2.13, 56.94, 47.66, 43.42, and 339 μC s −1 cm −2 , respectively, consistent with colorimetric methods. This method also allows the evaluation of nanozyme dependence on H 2 O 2 concentration, offering new insights into nanozyme properties and their potential applications in biosensing.