Dual-Modal Immunosensor Made with the Multifunction Nanobody for Fluorescent/Colorimetric Sensitive Detection of Aflatoxin B₁ in Maize

In recent years, dual-modal immunosensors based on synthetic nanomaterials have provided accurate and sensitive detection. However, preparation of nanomaterial probes can be time-consuming, laborious, and not limited to producing inactive and low-affinity antibodies. These challenges can be addressed through the multifunction nanobody without conjugation. In this study, a nanobody-enhanced green fluorescent (Nb26-EGFP) was novel produced with a satisfactory affinity and fluorescent properties. Then, a dual-modal fluorescent/colorimetric immunosensor was constructed using the Nb26-EGFP-gold nanoflowers (AuNFs) composite as a probe, to detect the aflatoxin B 1 (AFB 1 ). In the maize matrix, the proposed immunosensor showed high sensitivity with a limit of detection (LOD) of 0.0024 ng/mL and a visual LOD of 1 ng/mL, which is 20-fold and 325-fold compared with the Nb26-EGFP-based single-modal immunosensor and original nanobody Nb26-based immunoassay. The performance of the dual-modal assay was validated by a high-performance liquid chromatography method. The recoveries were between 83.19 and 108.85%, with the coefficients of variation below 9.43%, indicating satisfied accuracy and repeatability. Overall, the novel Nb26-EGFP could be used as the detection probe, and the dual-modal immunosensor could be used as a practical detection method for AFB 1 in real samples.