Breaking the sensitivity barrier in ELISA: A molecularly engineered peptide nanointerface achieves ultrasensitive...

Biosensing performance is often compromised by antibody activity loss caused by random immobilization, which remains a critical challenge in immunoassay development. Conventional trial-and-error strategies for interface design lack mechanistic insight, limiting the optimization of antibody orientation and function. Here, we address this gap by establishing a molecular simulation-driven rational design paradigm to construct optimal antibody-oriented interfaces for enhanced biosensing performance.

Molecular dynamics simulations revealed that the HWRGWVC heptapeptide mediates directional antibody immobilization via synergistic electrostatic pre-orientation (Arg/His residues) and hydrophobic interactions (Trp), with a binding energy of -41.17 kcal/mol. Guided by this mechanism, we developed an icELISA for zearalenone detection. Compared to random immobilization, the oriented approach increased antibody coupling efficiency to 84.3%, Fab exposure rate to 60.9 %, and affinity to 3.86 × 10⁸ M^-1, while reducing antibody consumption by 50 %. The assay achieved a detection limit of 0.006 μg/kg in grains (100-fold more sensitive than conventional ELISA), with recoveries of 91.1-101.9 %, CVs of 3.7-9.2 %, and stability over 150 days. Results from 10 natural samples correlated strongly with LC-MS/MS (R² > 0.95).

This work delivers a highly sensitive, simulation-informed platform for mycotoxin detection in grains, while establishing a generalizable framework for rational design of next-generation immunoassays. By overcoming antibody activity loss via oriented immobilization, the strategy enhances assay performance and reduces reagent consumption, offering broad utility in biosensing and food safety applications.