Genetic engineering-powered dual-mode lateral flow immunosensor for colorimetric and fluorescent detection of ochratoxin A in pepper

Ochratoxin A (OTA) poses significant risks to both environment and human health, necessitating the development of rapid and sensitive detection methods. Lateral flow immunosensors (LFIs) typically use monoclonal antibodies and artificial antigens for mycotoxin detection; however, the preparation of these components is time-intensive, laborious, costly, and environmentally harmful. Herein, we developed an innovative genetic engineering-powered colorimetric/fluorescent dual-mode LFI (CM/FL-dLFI) using nanobodies and the mimotope peptide (MP) of OTA. Initially, a heptameric bifunctional fusion (sfGFP-C4bpα-Y4) containing the superfolder green fluorescent protein, the C4-binding protein α-chain, and the MP Y4 was constructed. The fusion was subsequently labeled on gold nanoflowers (AuNFs) to create the AuNFs@sfGFP-C4bpα-Y4 probe, which can be captured by the nanobody heptamer (Nb-C4bpα) and provides colorimetric/fluorescent signals. The optimized CM/FL-dLFI achieved a colorimetric limit of detection (LOD) of 0.01 ng/mL and a fluorescent LOD of 0.05 ng/mL. It exhibited high selectivity for OTA, good recovery rates of 82.85–102.81 % with relative standard deviations not exceeding 14 %, and excellent long-term stability. Moreover, it showed strong correlation with high-performance liquid chromatography in the analysis of 11 real pepper samples. Therefore, this study highlights the promising application potential of nanobodies and MPs in developing an economical and eco-friendly LFI for OTA detection in pepper.