Natural Enzyme-Inspired Design of the Single-Atom Cu Nanozyme as Dual-Enzyme Mimics for Distinguishing Total Antioxidant Capacity and the Ascorbic Acid Level

Although various oxidase mimetic or peroxidase (POD) mimetic nanozymes have been extensively studied, their poor substrate selectivity significantly inhibits their practical applications. Nanozymes with specific biomolecules as substrates, especially ascorbic acid oxidase (AAO) mimetic nanozymes with ascorbic acid (AA) as a substrate, have scarcely been studied. Herein, inspired by the multi-Cu atom sites and the redox electron transfer pathway of Cu²⁺/Cu⁺ in the natural AAO, atomically dispersed Cu sites immobilized on N-doped porous carbon (Cu-N/C) are artificially designed to simulate the function of natural AAO. Compared with their natural counterparts, the Cu-N/C catalysts exhibited higher catalytic efficiency and superior stability. Combined theoretical calculation and experimental characterizations reveal that the Cu-N/C nanozymes could catalyze the AA oxidation through a 2e^- oxygen reduction pathway with H₂O₂ as the product. Moreover, the Cu-N/C nanozymes also possess high POD activity. As a proof-of-concept application, Cu-N/C can simultaneously realize AA detection in fluorescent mode based on its AAO activity and total antioxidant capacity detection in colorimetric mode utilizing its POD activity.