Fabrication of a robust and ultra-sensitive SERS platform via photoaffinity labeling-mediated oriented and covalent antibody immobilization for aflatoxin B1 biosensing.

To address the critical need for robust and ultrasensitive detection of aflatoxin B1 (AFB1), we developed a surface-enhanced Raman scattering (SERS) immunosensor featuring a precise, covalent, and uniformly oriented bio-interface. This was achieved via a genetically engineered photoaffinity labeling strategy. Specifically, an engineered Fc-binding protein-incorporating a photoreactive unnatural amino acid and a terminal cysteine tag-was utilized to site-specifically label the antibody Fc region under mild UV irradiation. The resulting thiol-modified antibody was then directionally anchored onto a gold nanoparticle substrate via robust Au-S self-assembly, ensuring an optimal "Fab-up" configuration. Relying on this highly efficient architecture, an indirect competitive SERS immunoassay was constructed. The platform exhibited an exceptional limit of detection (LOD) of 0.2 pg mL-1 and a broad linear dynamic range spanning from 0.001 to 10 ng mL-1, alongside excellent specificity against co-occurring mycotoxins. Applied to real peanut oil extracts, the assay demonstrated highly reliable recoveries (99.4%-106.8%, RSD <6%) that perfectly correlated with standard LC-MS/MS validation. This work provides a highly viable analytical tool for trace food safety surveillance and establishes a universal paradigm for precision-engineering high-performance optical biosensing interfaces.