Optimization of the diaminobenzidine substrate composition improves the performance of peroxidase-like Prussian Blue nanozymes in colorimetric assays

One of the emerging trends in modern analytical and bioanalytical chemistry is the substitution of enzyme labels, such as horseradish peroxidase, with nanozymes — nanoparticles that possess enzyme-like catalytic activity. Since enzymes and nanozymes typically have different catalytic mechanisms, it is expected that the optimal reaction conditions will also differ. The objective of this paper was to optimize the diaminobenzidine (DAB) chromogenic substrate to enhance the performance of Prussian Blue nanozymes in immunostaining applications. We elucidated that enhancers (such as imidazole), which are typically added to commercial DAB substrate solutions to boost the color signal generated by peroxidase, can suppress the activity of nanozymes. This issue can be resolved by optimizing a nanozyme-specific substrate buffer composition. More specifically, we demonstrated that buffers such as citrate, MES, HEPES, and TRIS containing 1.5-2 M NaCl or NH4Cl substantially increase DAB oxidation by Prussian Blue and provide a higher signal than commercial DAB formulations. Moreover, the composition of the substrate solution (type of buffer, ionic strength, additives) affects signal intensity to the same degree as buffer pH. Tuning the substrate buffer composition is, therefore, a simple and efficient way of signal amplification in nanozyme-based assays. Optimized DAB substrate formulations were applied to Prussian Blue-based tissue staining, western blotting assay of immunoglobulins, and dot blot assay of antibodies against SARS-CoV-2.