Nanobodies in Immunoanalysis: From Molecular Engineering to Advanced Detection Strategies for Foodborne Natural Toxins.

Natural toxins in food pose significant health risks, such as cancer, deformities, etc., demanding efficient and accurate surveillance techniques. Immunoassays become reliable alternatives to traditional chromatographic technologies for rapid, routine, and large-scale detection of natural toxins, but their performance depends highly on antibodies used, which are limited by stability issues and productions challenge. Nanobodies (Nbs) overcome these limitations, enhancing immunoassay sensitivity, specificity, and practicality for low-abundance toxin detection due to their good penetration/binding ability, inherent stability, and scalable production. This review offers a comprehensive, bottom-to-top overview of emerging Nb-based immunoassays, from foundational Nbs to advanced applications in natural toxin detection. The bioactivity of Nbs was summarized, focusing on their structural and functional characteristics critical for advancing immunoassays. Subsequently, the advanced methods for their screening, expression, and purification were outlined. Based on the natural toxins they target, both conventional and innovative Nb-based immunoassays are reviewed, in which corresponding Nbs modification strategies are highlighted for improving the affinity and sensitivity. The current challenges, potential strategies, and future opportunities for developing and applying Nb-based immunoassays are also discussed in natural toxin detection. This review, bridging fundamental Nb advances and immunoassay innovation, offers valuable perspectives to guide future development of Nb-based detection platforms.