Bioorthogonal modular assembly of functionalized nanobodies: A universal SNAP-click strategy for developing multiplex...

This study introduces a novel bioorthogonal modification strategy for nanobodies, leveraging SNAP-tag-mediated site-specific conjugation and click-chemistry to significantly enhance their applicability in food safety monitoring and biomedical applications. By employing the Nb10E-SNAP fusion protein alongside azide-labeled benzylguanine (N₃-BG) and alkyne/DBCO-functionalized moieties (Biotin-Alkyne, FITC-Alkyne, DBCO-ssDNA, and DBCO-quantum dots), we have successfully engineered four distinct Aflatoxin B1 (AFB1) immunoassay methods: BA-CLIA (Biotin-Avidin Chemiluminescence Immunoassay), one-step FLIA (Nanobody-based Fluorescent Immunoassay), Nb-RCA (Nb-based Rolling Circle Amplification), and Nb-LFIA (Nb-based Lateral Flow Immunoassay). These methods have demonstrated markedly improved sensitivity over traditional Nb-SNAP-based ELISA (LOD: 0.26 ng/mL), with detection limits of 0.21 ng/mL (BA-CLIA), 0.10 ng/mL (one-step FLIA), 0.0095 ng/mL (Nb-RCA), and 0.64 ng/mL (Nb-LFIA), respectively. The rolling circle amplification (RCA)-enhanced Nb-RCA platform, in particular, showcased a 27-fold increase in sensitivity compared to conventional ELISA, underscoring the efficacy and adaptability of our bioorthogonal modification approach. Additionally, the analytical performance of Nb-RCA and Nb-LFIA was assessed in oat and milk samples, yielding recovery rates of 85.20% to 124.60% and 82.71% to 97.83%, respectively, indicating robust performance in complex food matrices. This research has culminated in the development of a new, efficient, and accessible method for the modular functionalization of nanobodies. As the scope of nanobody applications continues to grow, we anticipate that this strategy will offer a straightforward and practical solution for harnessing the potential of a wider array of nanobodies, thereby facilitating more sophisticated applications across diverse fields.