Self-Assembly Multivalent Fluorescence-Nanobody Coupled Multifunctional Nanomaterial with Colorimetric Fluorescence and Photothermal to Enhance Immunochromatographic Assay

The multimodal lateral flow immunoassay (LFIA) has provided accurate and reliable results for fast and immediate detection. Nonetheless, multimodal LFIA remains challenging to develop biosensors with high sensitivity and tolerance to matrix interference in agro-food. In this study, we developed a self-assembled multivalent fluorescence-nanobody (Nb26-EGFP-H6) with 16.5-fold and 30-fold higher affinity and sensitivity than a monovalent nanobody (Nb26). Based on the Nb26-EGFP-H6, we synthesized enhanced immune-probes Zn-CN@Nb26-EGFP-H6 by pyrolyzing and oxidizing an imidazolating zeolite framework-8 (ZIF-8) to obtain photothermal metal-carbon nanomaterials (Zn-CN) for immobilizing Nb26-EGFP-H6. The rough and porous structure of Zn-CN with a large surface area facilitates the enrichment and immobilization of antibodies. A trimodal lateral flow immunoassay (tLFIA) with colorimetric, fluorescent, and photothermal triple signal outputs was constructed for the detection of aflatoxin B₁ (AFB₁) in maize. Attractively, the Zn-CN-based tLFIA's multiplex guarantees accurate and sensitive detection of AFB₁, with triple signal detection limits of 0.0012 ng/mL (colorimetric signals), 0.0094 ng/mL (fluorescent signals), and 0.252 ng/mL (photothermal signals). The sensitivity of the trimode immunosensor was 628-fold and 42-fold higher than that of the original Nb26-based ELISA (IC₅₀) and the unimodal LFIA (LOD). This work provides an idea for constructing a sensitive, tolerant matrix and efficient and accurate analytical platform for rapidly detecting AFB₁ in food.