Boronate-mediated antibody orientation: a molecular simulation-guided strategy for ultra-sensitive lateral flow immunoassays.

The random immobilization of antibodies on lateral flow immunoassay (LFIA) strips remains a major factor limiting assay sensitivity and consistency. To address this, we report a boronate affinity-based strategy for site-specific antibody orientation via covalent reaction between boronic acid and cis-diol groups on Fc-region glycans. Molecular dynamics simulations provided mechanistic support for this design. The boronate-antibody configuration demonstrated the most favorable binding energy (- 153.93 kJ/mol) and the fastest conformational stabilization (< 40 ns), suggesting a stabilization process involving initial electrostatic guidance followed by hydrogen-bond and covalent bond formation. Experimentally, this oriented immobilization achieved an antibody conjugation efficiency of 99.92%, a Fab exposure rate of 47.52% and significantly improved functional activity, with an affinity constant of 0.93 × 10⁸ M⁻¹, outperforming conventional passive adsorption. Applied to the detection of zearalenone (ZEN) in corn, the oriented LFIA showed a dramatic sensitivity enhancement. The limit of detection reached 0.049 µg/kg, 200-fold higher than traditional colloidal gold LFIA. The visual cut-off value was 0.5 µg/kg. Validation with spiked and naturally contaminated samples yielded recoveries of 84.7%-103.4% (CV: 7.1%-12.6%) and excellent correlation (R