Site-specific conjugation of nanobodies mediated by SNAP enhances the performance of nanobody-based lateral flow immunoassay

The preparation of nanoparticle–nanobody (Nb) conjugates with high stability and affinity remains challenging in lateral flow immunoassay (LFIA) development. Herein, we employed a SNAP-O(6)-benzylguanine (BG) system to achieve site-specific conjugation of Nb-snap fusion proteins with BG-coated fluorescent microspheres (FMs), yielding FM/BG@Nb-snap probes with enhanced stability and bioactivity. The FM/BG@Nb-snap-LFIA enabled quantitative ochratoxin A (OTA) detection within 10 min, with a limit of detection of 0.06 ng/mL, representing 3.5-fold and 39.17-fold improvements over conventional covalent coupling and colloidal gold methods, respectively. Recovery rates for OTA detection in food samples ranged from 93.08% to 111.55%. Overall, this study offers a promising strategy for the design and preparation of Nb-based probes to enhance LFIA performance.