Resolving the issue of non-specific substrate oxidation in nanozyme-assisted colorimetric assays

Nanoparticles exhibiting peroxidase-like activity (nanozymes) are emerging as promising alternatives to enzyme labels in colorimetric assays, including ELISA. Unlike natural peroxidases, nanozymes demonstrate distinct mechanisms for oxidizing chromogenic substrates such as 3,3',5,5'-tetramethylbenzidine (TMB), prompting the use of custom substrate formulations in contemporary research. However, analysis of the literature reveals that such compositions often yield abnormally high background signals due to non-specific oxidation of TMB, likely catalyzed by trace metal ions present in assay buffers. This phenomenon narrows the dynamic range, elevates limits of detection (LOD), and restricts the use of higher substrate concentrations that could otherwise enhance assay sensitivity. In this study, we identify the widespread use of sodium acetate buffer as a principal contributor to non-specific oxidation, owing to its limited chelating capacity. Statistical analysis demonstrates that lower pH, elevated TMB concentration, and, to a lesser extent, increased buffer molarity and hydrogen peroxide concentration are the most significant factors promoting non-specific reactions. Additional variables influencing the background include reagent purity and organic co-solvent content. Employing platinum nanozymes as a model system, we propose two effective strategies to mitigate non-specific oxidation: (i) substitution of acetate buffer with citrate buffer and (ii) supplementation of acetate buffer with chelating agents. These approaches reduce LOD by 3-12-fold while maintaining low background signals even under adverse conditions (high TMB concentration and low pH).