Fe–N 3 S Single-Atom Nanozyme with Asymmetric Coordination for Ultra-Low-Background Colorimetric Immunoassays

Abstract

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–N 3 S single-atom nanozyme with a unique peroxidase (POD) catalytic pathway. Specifically, the Fe–N 3 S nanozyme exhibited high POD-like activity (26.82 U mg –1 ) and low oxidase (OXD)-like activity (0.018 U mg –1 ), whereas Fe–N 4 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–N 4, 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–N 3 S 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.

The authors' abstract, as published at the source. Analytical Chemistry, 2026 · DOI ↗