Flower-like FeMoO₄ nanoparticles as a peroxidase mimic for sensitive colorimetric immunoassay of CEA

Early and accessible detection of tumor biomarkers requires robust colorimetric readouts that can operate in complex matrices. We report flower-like hierarchical FeMoO₄ nanoparticles functioning as a peroxidase-like nanozyme and enable a cascade immunoassay for carcinoembryonic antigen. The nanoparticles were obtained by a solvothermal route and exhibit a single crystalline FeMoO₄ phase with uniform Fe, Mo, and O distributions, mixed Fe³⁺ and Fe²⁺ states, and a measurable fraction of surface non-lattice oxygen species (O_ads, including hydroxyl-related oxygen and adsorbed/weakly bound oxygen). These features support efficient H₂O₂ activation and TMB oxidation. FeMoO₄ was integrated with a glucose oxidase (GOx)-based enzyme signal reporter, in which GOx and the detection antibody (Ab2) were co-immobilized on gold nanoparticles (AuNPs) to form GOx-AuNP-Ab2 conjugate. In a sandwich immunoassay format, the captured enzyme-labeled conjugate generates H₂O₂ in proportion to the antigen concentration, which is subsequently converted by FeMoO₄ into a stable colorimetric signal at 652 nm. Reaction conditions were optimized for both the chromogenic branch and the enzymatic branch, and kinetic analysis gave Michaelis-Menten behavior with K_m values of 0.78 mM for H₂O₂ and 0.40 mM for TMB. Radical scavenging identified superoxide and hydroxyl radicals as the main active species, with singlet oxygen as a minor contributor. The CEA assay achieved a linear range of 0.1 to 60 ng mL^-1 and a limit of detection of 63 pg mL^-1, with a one month retention of 93% of the initial absorbance. Results for six clinical serum samples showed good agreement with a commercial ELISA at the 95% confidence level, and one CEA-negative serum was included as a negative control. These findings establish FeMoO₄ as a stable and manufacturable nanozyme platform for colorimetric immunoassays.