A novel triple-display platform based on MS2 Virus-Like Particles for ultra-sensitive immunoassay of Aflatoxin B₁

Aflatoxin B₁ (AFB₁), a Group I carcinogen, poses severe health threats in food and environmental matrices, which requires ultra-sensitive monitoring tools. To address this, we engineered MS2 Virus-Like Particles (VLPs) via a novel triple-display strategy integrating: (i) surface-exposed nanobody N26 (fused to the A-protein) for specific recognition; (ii) high-density biotinylation (∼90 molecules/VLP, via Avi Tag insertion) for signal amplification; and (iii) C-terminal His-tags on coat protein dimers conferring organic solvent tolerance and simplified purification. The resulting Biotin-His&N26@MS2 VLP facilitates multivalent biotin-streptavidin interactions, dramatically amplifying detection signals. In indirect competitive ELISA, this platform achieved an ultra-low limit of detection (LoD) of 1 pg/mL for AFB₁ - representing a >1000-fold improvement over monovalent biotin of nanobody N26 - with a broad linear range (5.3-34,400 ng/mL). Validation using spiked maize samples demonstrated high accuracy (recoveries: 84-128 %) and excellent correlation with LC-MS. Critically, the VLP retained full functionality in 20 % methanol, confirming its utility for complex matrices requiring organic solvent extraction. This work establishes a robust, organic solvent-tolerant VLP strategy enabling high-throughput surveillance of hazardous mycotoxins in agricultural and environmental settings.