Electrochemical tropomyosin allergen immunosensor for complex food matrix analysis

A shrimp tropomyosin (TPM) immunosensor has been developed and optimized to detect trace amounts of shrimp (in the ppm range), based on a combination of an amperometric transduction, magnetic particles and disposable screen-printed electrodes. The approach is based on the implementation of a sandwich immunoassay format on the surface of magnetic beads and their coupling onto disposable screen-printed electrodes to finally register the amperometric response at -200 mV vs. Ag pseudo-reference electrode, using H₂O₂ as enzymatic substrate and hydroquinone as redox mediator. The use of carboxyl-functionalized magnetic microbeads (MBs) and in-house made magnetic nanoparticles (MNPs) as solid supports have been evaluated and compared. Our experimental results confirm that the use of MBs, in addition to simplifying the test protocol, improves the resulting sensitivity, so they were selected for the implementation of the immunosensor. In the optimized experimental conditions, the developed immunosensor offered a LOD of 47 pg mL^-1 for amperometric determination of shrimp TPM standards and great selectivity against TPM from other sources, thus allowing differentiation between crustaceans (shrimp) and mollusks (squid). Applicability studies demonstrated successful determination both in crude and cooked samples using very simple protocols. Additionally, processed foods based on fish and mollusks that could potentially include crustaceans in their composition have been analyzed using the sensor and compared to the declared ingredients. The sensitivity and specificity showed by the sensor in the analysis of heterogeneous food samples without a previous purification or enrichment stage, also outperforms existing solutions in terms of time and cost effectiveness and permits its direct and smooth implementation in the food industry for routine allergen analyses.