Nanobody-Based Lateral Flow Assays: Applications, Innovations, Challenges, and Future Directions.

Nanobody-based lateral flow assays (LFAs) are an emerging class of point-of-care diagnostics that combine enhanced thermal stability, target affinity comparable to or in some cases exceeding that of conventional antibodies, and low-cost recombinant production of nanobodies with the versatility of conventional antibody-based detection formats. This review examines recent progress in nanobody engineering, assay architecture, and signal amplification strategies that improve analytical sensitivity, specificity, and multiplexing potential. Despite these advances, several technical and translational challenges persist, including broad analyte compatibility, difficulties in optimal nanobody pair selection, scalability of expression systems, and the need for robust and reproducible conjugation chemistries. The growing integration of computational modeling and AI-driven nanobody design has accelerated the discovery and optimization pipelines, yet standardized frameworks for validation and benchmarking remain scarce. Addressing these limitations will be important for the further clinical translation and commercial deployment of nanobody-based LFAs across diverse applications in infectious disease diagnostics, food safety testing, and environmental monitoring.