A universal one-pot assay strategy based on bio-inorganic cascade catalysts for different analytes by changing pH-dependent activity of enzymes on enzyme mimics

Abstract In general, peroxidase mimics show optimal activity at acidic pH, while many enzymes work at physiological pH. To realize one-pot colorimetric detection based on enzyme-nanozyme-coupled system, the usual strategy is to change the pH-dependent activity of nanozymes, which is difficult for most of nanozymes. Unlike common strategies, we changed the pH-dependent activity of enzymes rather than nanozymes. First, we facilely synthesized Co 3 O 4 magnetic peroxidase mimics with uniform size, good dispersibility and superparamagnetism by using protein as templates at mild condition. The as-prepared Co 3 O 4 magnetic nanoparticles (MNPs) were covered with the layers of protein, facilitating the further functionalization with enzymes. Exhilaratingly, we discovered that the immobilization on Co 3 O 4 MNPs could regulated pH-dependent activity of enzymes, such as glucose oxidase (GOx) and glucoamylase (GA). Based on the overlapped pH range between peroxidase-like activity of Co 3 O 4 MNPs and catalytic activity of GOx, the hybrid cascade catalysts were employed for a one-pot glucose detection, exhibiting wide detection range and good selectivity. Furthermore, GA and GOx were co-mobilized on Co 3 O 4 MNPs to realize the one-pot colorimetric starch detection, confirming the universality of the proposed strategy. As an additional bonus, hybrid cascade catalysts could be expediently separated from the reaction solution under the magnetic field in order to rapidly terminate the reaction and recycle enzymes. This work would open an avenue for the various one-pot enzyme-nanozyme-coupled assay.