Biosynthetic small molecule antigens mimics medicated lateral flow immunoassay for mycotoxin Fumonisin B₁ using nanobody fusion proteins

Lateral flow immunoassays (LFAs) are widely used in point-of-care testing (POCT) for detecting small molecules. However, their application is often hindered by the complex synthesis of traditional chemically synthesized antigens. Nanobody-based coating antigen mimics have shown excellent analytical performance in various immunoassay platforms, but their application in LFAs still faces challenges. Here, we demonstrate the use of nanobody fusion proteins as antigen mimics in the construction of LFAs for detecting small molecules. Anti-idiotypic nanobody (B26) specific to Fumonisin B₁ (FB₁) was selected as a case study, and the maltose binding protein (MBP)-fused B26 showed optimal performance. Adsorption studies revealed that MBP-B26 showed an approximately 8-fold enhancement in maximum equilibrium adsorption capacity compared to unmodified B26 on nitrocellulose filter (NC) membranes in LFAs, and nearly 1.5-fold increase compared to chemically synthesized antigens. Molecular dynamics simulations also indicated a 28.7 % increase in non-bonding interaction energies. The MBP-B26-based LFAs detection method for FB₁ demonstrated high sensitivity (1.76 μg/kg) and more environmentally friendly compared to chemically synthesized antigens. This study establishes a theoretical framework and provides models for the use of nanobody fusion proteins-based LFAs, facilitating broader application in detecting small molecules in POCT.