Nanoparticle-free dualmodal lateral flow immunoassay for sensitive detection of aflatoxin B1 based on "four-in-one" multifunctional nanobody.

BACKGROUND: Aflatoxin B1 (AFB1), a potent mycotoxin prevalent in food commodities, poses severe health threats requiring rapid and ultra-sensitive on-site detection. Lateral flow immunoassays (LFIAs) have emerged as a promising solution due to their simplicity, low cost, and rapid result readout. However, conventional LFIAs relying on nanomaterial-labeled antibodies face challenges such as complex fabrication, limited stability, and notably reduced analytical sensitivity when using nanobodies. It is thus essential to develop a streamlined and robust LFIA platform that overcomes the inherent constraints of conventional nanobody-based assays while retaining their practical advantages for point-of-care applications.

RESULTS: Here, we rationally engineered a novel multifunctional fusion protein designated stGFP-ALP-Nb, incorporating StayGold fluorescent protein (stGFP), alkaline phosphatase (ALP), and an Avi-tagged nanobody (Nb) through structure-guided design. Functional validation confirms the integrated system's activity. Based on this multifunctional nanobody fusion protein, we present a nanoparticle-free, dual-modal LFIA, which integrates green fluorescence, enzymatic activity, antigen recognition, and streptavidin-binding within a single construct. This "four-in-one" probe enables simultaneous fluorescence and chemiluminescence readouts without external labeling. The developed assay achieves a limit of detection (LOD) of 0.51 pg/mL (range: 4.7-57290.99 pg/mL) in fluorescence mode and 0.69 pg/mL (range: 3.28-4886.42 pg/mL) in chemiluminescence mode, and from sample preparation to result readout, the entire assay is completed within 30 min. Validated in spiked food samples, such as millet, quinoa, and rice, the method shows excellent specificity, recovery, and reproducibility, confirming robust analytical performance for practical applications.

SIGNIFICANCE: This design enables a nanoparticle-free dual-modal LFIA that eliminates the need for complex nanomaterial conjugation while substantially enhancing detection sensitivity. The platform offers a simplified, eco-friendly, and highly sensitive analytical system that serves not only as a robust tool for point-of-care mycotoxin monitoring but also establishes a versatile framework adaptable to nanobody-based biosensing applications in food safety, while simultaneously expanding the capabilities of rapid detection technologies.