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 IC₅₀ 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.