Colorimetric and surface-enhanced Raman scattering dual-mode lateral flow immunosensor using phage-displayed shark nanobody for the detection of crustacean allergen tropomyosin

Tropomyosin (TM) is the primary allergenic protein responsible for crustacean food allergies, and thus sensitive and rapid methods are required for the screening of crustacean TM in food. In this study, using the phage-displayed shark nanobody (PSN) as a multifunctional biomaterial, we developed a colorimetric and surface-enhanced Raman scattering dual-mode lateral flow immunosensor (CM/SERS-LFI) for competitive detection of crustacean TM. The SERS tag AuMBA@AgNPs with the Raman signal molecule 4-mercaptobenzoic acid (4-MBA) was prepared and immobilized on the PSN to construct the immunoprobe AuMBA@Ag-PSN. The probe can identify free TM that competes with TM on the T-line, and the optimized CM/SERS-LFI enables quantitative analysis of TM using the probe with a limit of detection (LOD) of 0.0026 μg/mL (SERS mode) and 0.0057 μg/mL (colorimetric mode), respectively. Additionally, it can implement a qualitative analysis by the naked eye with a visual LOD of 0.01 μg/mL. The CM/SERS-LFI exhibited excellent performance in the tests of selectivity, accuracy, precision, and stability. Moreover, the method's effectiveness in the analysis of real samples was confirmed by a commercial ELISA kit. Therefore, the developed CM/SERS-LFI was demonstrated to be a powerful and reliable tool for the rapid and sensitive detection of crustacean TM in food.