The optimization of gold nanoparticles–horseradish peroxidase as peroxidase mimic using central composite design for the detection of hydrogen peroxide

The oxidizing agent, hydrogen peroxide (H₂O₂), which is a part of reactive oxygen species (ROS) is well-known to contribute to oxidative stress-induced damage to biological molecules. An excess of free radicals can harm health and is associated with human diseases. Gold nanotechnology, a highly relevant nanomaterial, has been utilized as a new material in advanced sensor detection. In this study, colorimetric methods based on peroxidase enzymes were developed for measuring H₂O₂. The synthesized gold nanoparticles (AuNPs) showed a concentration of approximately 1.73 nM at a wavelength of 520 nm. The average diameter displayed a uniform size distribution, estimated at 18 nm, and an increase in the shell thickness of AuNPs-horseradish peroxidase (HRP) was observed in the TEM images. The AuNPs-HRP system demonstrated remarkable catalytic activity in the reaction of the chromogenic substrate tetramethylbenzidine (TMB) with H₂O₂, resulting in the production of an oxide product. The optimal conditions for the AuNPs-HRP system, as determined by central composite design (CCD), were a temperature of 25 °C and a pH of 7 within an 8 h period. A strong linear relationship was observed between different absorbance values and the H₂O₂ concentration, with a coefficient of determination of 0.9956. A portable platform was successfully used to determine H₂O₂ levels in beverages with recoveries ranging from 95.51% to 118.85%. These findings suggest that the AuNPs-HRP system could be applied to detect H₂O₂ in beverages.