Fe-N₃S Single-Atom Nanozyme with Asymmetric Coordination for Ultra-Low-Background Colorimetric Immunoassays

Although nanocatalysts have advanced highly sensitive immunoassays for low-abundance proteins, the nonspecific catalytic reactions cause high background signals and notable false-positive issues, hindering their further application. Herein, through an asymmetric coordination field modulation strategy, we rationally designed and synthesized an Fe-N₃S single-atom nanozyme with a unique peroxidase (POD) catalytic pathway. Specifically, the Fe-N₃S nanozyme exhibited high POD-like activity (26.82 U mg^-1) and low oxidase (OXD)-like activity (0.018 U mg^-1), whereas Fe-N₄ nanozymes showed the opposite trend (7.68 U mg^-1 for POD-like, 0.19 U mg^-1 for OXD-like). Density functional theory (DFT) calculations revealed that the introduction of sulfur disrupts the symmetric octahedral crystal field of Fe-N₄, resulting in specific repulsion toward the key intermediate OH* species. This was consequently manifested as a reduction in the energy barrier at the rate-determining step along the POD pathway. A low-background, highly specific immunochromatographic sensor for cluster of differentiation 40 ligand (CD40L) detection was developed based on a cascade catalytic reaction involving nanozymes and native enzymes. Immunosensors employing the Fe-N₃S nanozyme as chromogenic probes exhibited a lower limit of detection (0.69 pg mL^-1) and a satisfactory dynamic response range (0.001-1000 ng mL^-1), accompanied by good specificity and stability. This work opens new avenues for developing portable colorimetric immunosensing methods with low background interference.