Unveiling Generally‐ignored Co‐substrate Effect of Catalase‐inherent Peroxidase Mimic for Self‐verifiable Detection of High‐concentration Hydrogen Peroxide

Abstract One nanoparticle possessing both peroxidase (POD) and catalase (CAT) activities is a prevalent co‐substrate nanozyme system, distinct from the extensively researched cascade nanozyme system. During the sensing of hydrogen peroxide by POD, the impact of CAT is actually ignored in most studies. In this study, the CAT effect on hydrogen peroxide detection is thoroughly investigated based on POD catalysis by finely tuning the relative activity of POD and CAT. It is discovered that the CAT effect can be changed by delaying the injection of chromogenic substrate after adding hydrogen peroxide and that the linear range grows with the delayed time. Then, a theoretical mechanism showed that the time‐delay mediated CAT effect magnification does not change the Vmax, but it causes Km to linearly increase with delayed time, consistent with the experiment results. Furthermore, the detection of high concentrations of hydrogen peroxide is successfully realized in contact lens care solutions by utilizing time‐delay‐mediated POD/CAT nanozyme. On the other hand, its linear range‐tunable characteristic is used to produce multiple standard curves, then enabled self‐verifying hydrogen peroxide detection. Overall, this work investigates the role of CAT in CAT‐inherent POD nanozymes both theoretically and experimentally, and confirms POD/CAT nanozyme's priority in developing high‐performance sensors.