Linker-optimized bivalent nanobodies combined with photothermal gold liposome nanocomposites for enhanced dual-mode...

Lateral flow immunoassay (LFIA) has been widely used for rapid on-site analysis because of its simplicity, portability, and low cost. However, its performance in competitive assays, particularly for small molecule toxins, remains limited, as the properties of antibodies and the reliance on single-mode signal often lead to insufficient sensitivity and reliability. To address this challenge, we developed an enhanced colorimetric-photothermal LFIA by integrating linker-optimized bivalent nanobodies (BvNbs) and gold-liposome nanocomposites (Au-LNCs). Using tetrodotoxin (TTX) as a representative analyte, a series of BvNbs with different linker lengths were constructed. Molecular docking and molecular dynamics simulations further demonstrated that linker length modulates BvNbs detection performance by regulating conformational stability, local flexibility, and persistent interactions with free target molecules. In parallel, photothermal Au-LNCs were prepared, which exhibited broadband near-infrared absorption and a photothermal conversion efficiency as high as 78.06%. The resulting LFIA achieved a photothermal LOD of 1.89 ng/mL, corresponding to an approximately 10.7-fold improvement in sensitivity compared with conventional colloidal gold-based LFIA, while maintaining good reliability in real samples. Overall, this study provides mechanistic insight into the rational design of BvNbs and a promising strategy for improving LFIA performance.