Improving Nanozyme-BasedColorimetric Assays throughMedium Composition Optimization in Nanozyme-Substrate Reaction

Nanozymes, while promising alternatives to natural peroxidases in colorimetric assays, are often hindered by lower catalytic efficiencies. Although numerous approaches have been developed to improve signal intensity in nanozyme-based assays, optimization of the reaction medium in which the nanozyme interacts with the substrate remains a significantly underexplored area. The vast majority of studies rely on standard sodium acetate buffers or commercially sourced reagents optimized for horseradish peroxidase, neglecting the unique catalytic properties of different nanozymes. This work presents a systematic optimization of 3,3′,5,5′-tetramethylbenzidine (TMB)-based reaction medium composition for four common nanozymes: iron oxide, LaNiO₃, Mn-doped CeO₂, and platinum nanoparticles. Our findings reveal that while sodium acetate buffer is suitable for LaNiO₃, alternative buffers significantly enhance signal intensity (41–68%) for the other nanozymes. Further optimization of ionic strength, organic cosolvent type and concentration, and TMB/H₂O₂ concentrations yielded improvements in signal intensity, analytical sensitivity, and assay time. This study also identifies common pitfalls encountered during optimization of reaction conditions and proposes potential solutions. We posit that reaction medium should be a standard optimization step in the development of nanozyme-based assays, and the use of commercially sourced reagents with undisclosed compositions should be avoided.