Conformational Dynamics-Driven Engineering of Nanobody Biosensor for Enhanced Detection of Ochratoxin A

Nanobodies are promising recognition elements for food safety biosensors, yet the relationship between their conformational dynamics and ligand binding remains poorly understood. This study presents an integrated computational–experimental framework to elucidate conformational dynamics-function relationships and guide the engineering of nanobody-based detection systems for ochratoxin A (OTA), a prevalent mycotoxin in agricultural commodities. Using the anti-OTA nanobody NB28, we employed molecular dynamics simulations, ion mobility-mass spectrometry, and collision-induced unfolding assays to investigate the ligand recognition mechanisms. NB28 binds OTA through a tunnel-like mechanism, inducing pronounced conformational shifts toward extended states, validated experimentally through collision cross-sectional measurements and unfolding profiles. Leveraging these dynamic insights, we engineered a T80C variant exhibiting ∼1.8-fold enhanced detection sensitivity with reduced IC 50 values while preserving specificity. T80C demonstrated robust performance across cereal matrices, achieving detection limits well below the EU regulatory threshold of 5 μg/kg. This dynamics-driven approach provides a generalizable strategy for food safety biosensor development.