Hemoglobin-assisted, ZIF-derived porous bimetallic FeCo/N-C nanozyme with high oxidase-like activity for colorimetric sensing.

Oxidative stress caused by excessive reactive oxygen species (ROS) is related to many diseases, and the importance of accurate detection of antioxidants is emphasized. Ascorbic acid (AA), a representative antioxidant, is widely used to assess total antioxidant capacity. Herein, we report the rational design of a bimetallic FeCo/N-C nanozyme via pyrolysis of a hemoglobin (Hb)-doped ZIF-67@ZIF-8 precursor. The Hb precursor plays a dual role: it acts as a molecular porogen to produce hierarchical porous structure, and as a coordination modulator to facilitate the formation of highly dispersed metal sites, thereby preventing cobalt aggregation and enhancing mass transfer. Crucially, the synthesized FeCo/N-C nanozyme exhibits exceptional oxidase-like activity by catalyzing the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) via a singlet oxygen (¹O2)-dominated non-radical pathway, as verified by electron paramagnetic resonance (EPR) spectroscopy. Based on AA's reductive suppression of TMB oxidation, a sensitive and H2O2-free colorimetric sensing platform was developed for the quantification of AA. Under optimized conditions, the sensor displayed wide dual-linear ranges of 0.025-2.5 µM and 2.5-150 µM, a low detection limit of 0.006 µM, along with desired selectivity and stability. The method demonstrated practical applicability for AA quantification in body fluid samples, with satisfactory recoveries (98.14-104.36%) and good reproducibility (RSD < 3.06%). This work provides a biomolecule-assisted strategy for the synthesis of high-efficiency nanozyme, and highlights the advantage of non-free radical catalysis mechanisms in constructing robust biosensor for clinical diagnosis and antioxidant analysis.