A Cascade Enzyme System Integrating Peroxidase Mimic with Catalase for Linear Range Expansion of H2O2 Assay: A Mechanism and Application Study

Abstract Peroxidase (POD) Nanozyme‐based hydrogen peroxide (H 2 O 2 ) detection is popular, but hardly adapt to high concentration of H 2 O 2 owing to narrow linear range (LR) and low LR maximum. Here, a solution of combining POD and catalase (CAT) is raised to expand the LR of H 2 O 2 assay via decomposing part of H 2 O 2 . As a proof of concept, a cascade enzyme system (rGRC) is constructed by integrating ruthenium nanoparticles (RuNPs), CAT and graphene together. The rGRC‐based sensor does perform an expanded LR and higher LR maximum for H 2 O 2 detection. Meanwhile, it is confirmed that LR expansion is closely associated with apparent K m of rGRC, which is determined by the relative enzyme activity between CAT and POD both in theory and in experiment. At last, rGRC is successfully used to detect high concentration of H 2 O 2 (up to 10 m m ) in contact lens care solution, which performs higher assay accuracy (close to 100% recovery at 10 m m of H 2 O 2 ) than traditional POD nanozymes. This study brings up a kind of POD/CAT cascade enzyme system and provides a new concept for accurate and facile H 2 O 2 detection. Additionally, it replenishes a new enzyme‐substrate model of achieving the same pattern with competitive inhibition in enzyme reactions.