Enhancing Nanozyme-Based Colorimetric Assays by Optimizing Substrate Composition

Nanozymes, while promising alternatives to natural peroxidases in colorimetric assays, are often hindered by lower catalytic efficiencies. While various strategies exist to enhance signal intensity in nanozyme-based assays, substrate optimization remains largely underexplored. The vast majority of studies rely on standard sodium acetate buffers or commercially-sourced substrates 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 substrate compositions 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 co-solvent 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 substrate optimization and proposes potential solutions. We posit that substrate composition should be a standard optimization step in the development of nanozyme-based assays, and the use of commercially-sourced substrates with undisclosed compositions should be avoided.