Improving Nanozyme-Based Colorimetric Assays through Medium Composition Optimization in Nanozyme-Substrate Reaction

Area of Science:

• Biochemistry
• Nanotechnology
• Analytical Chemistry

Background:

• Nanozymes offer a promising alternative to natural peroxidases in colorimetric assays.
• However, their catalytic efficiency is often limited, hindering assay performance.
• Optimizing the reaction medium is crucial but underexplored for nanozyme applications.

Purpose of the Study:

• To systematically optimize the reaction medium for 3,3',5,5'-tetramethylbenzidine (TMB)-based colorimetric assays using four common nanozymes.
• To investigate the impact of buffer composition, ionic strength, cosolvents, and substrate concentrations on nanozyme activity.
• To identify common challenges in reaction condition optimization and propose solutions.

Main Methods:

• Evaluated four nanozymes: iron oxide, LaNiO3, Mn-doped CeO2, and platinum nanoparticles.
• Systematically varied buffer types, ionic strength, organic cosolvents, and substrate (TMB/H2O2) concentrations.
• Assessed signal intensity, analytical sensitivity, and assay time.

Main Results:

• Standard sodium acetate buffer is optimal only for LaNiO3 nanozymes.
• Alternative buffers enhanced signal intensity by 41-68% for iron oxide, Mn-doped CeO2, and platinum nanoparticles.
• Optimized conditions improved signal intensity, sensitivity, and reduced assay time.

Conclusions:

• Reaction medium optimization is a critical, yet often overlooked, step for nanozyme-based assays.
• Tailoring the reaction environment significantly enhances nanozyme catalytic efficiency and assay performance.
• Avoid using commercially sourced reagents with undisclosed compositions for reliable results.