Flower-like Fe3S4 nanozymes with peroxidase-like activity for sensitive colorimetric immunodetection of carcinoembryonic antigen.

Flower-like Fe3S4 nanosheets were synthesized via a one-step solvothermal method and employed as peroxidase-mimicking nanozymes in a colorimetric immunoassay for the sensitive detection of carcinoembryonic antigen (CEA). Comprehensive structural and chemical characterizations confirmed the successful synthesis and surface modification of the Fe3S4 nanosheets, enabling efficient antibody conjugation. The nanozymes demonstrated significant peroxidase-mimicking behavior by facilitating the hydrogen peroxide (H2O2)-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), where hydroxyl radicals (•OH) were confirmed to be the dominant reactive intermediates. Kinetic studies revealed low Michaelis-Menten constants for both TMB and H2O2, indicating high substrate affinity and excellent catalytic efficiency. Under optimized conditions, the developed immunoassay showed a linear response in the CEA concentration range 0.05-50 ng/mL, with a detection limit of 82 pg/mL, which is well below the commonly used clinical cutoff for CEA (5.0 ng/mL). The assay exhibited good reproducibility (RSD = 4.2%), retained 93% of its activity after one week of storage, and showed strong agreement with commercial ELISA kits in clinical serum samples. The novelty of this work lies in the application-oriented design of flower-like Fe3S4 nanosheets via a simple one-step solvothermal route, delivering a sensitive and practical colorimetric immunoassay with robust stability and reproducibility. Taken together, these results establish a robust and cost-effective nanozyme platform with promising potential for clinical diagnostics and point-of-care tumor biomarker detection.