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.

Amperometric magnetoimmunosensor for ErbB2 breast cancer biomarker determination in human serum, cell lysates and intact breast cancer cells.

A highly sensitive amperometric magnetoimmunosensor for the determination of ErbB2 protein, a well-known biomarker related to high-impact high-incidence diseases such as breast cancer, is described. A sandwich format involving the covalent immobilization of a specific capture antibody onto magnetic beads (MBs) and incubation of the modified MBs with a mixture solution of the antigen and a HRP-labeled detector antibody was used. The resulting modified MBs were captured on the surface of a disposable screen-printed carbon electrode (SPCE) and the amperometric responses at -0.20 V were measured. This ErbB2 magnetoimmunosensor exhibited a very low detection limit of 26 pg mL(-1) far below the established cut-off for this biomarker (15 ng mL(-1)) and was successfully applied to the quantitation of ErbB2 in human serum and cell lysates samples without any matrix effect. In addition, the developed assay allowed the assessment of ErbB2 status directly in intact breast cancer cells. The results correlated well with those obtained with a commercial ELISA method, thus demonstrating that the new magnetoimmunosensing platform offers a truthful and useful analytical tool to be easily applied in breast cancer diagnosis through either ErbB2 protein determination or breast cancer cell status detection.

Amperometric magnetoimmunoassay for the direct detection of tumor necrosis factor alpha biomarker in human serum.

An amperometric immunoassay for the determination of tumor necrosis factor alpha (TNFα) protein biomarker in human serum based on the use of magnetic microbeads (MBs) and disposable screen-printed carbon electrodes (SPCEs) has been developed. The specifically modified microbeads were magnetically captured on the working electrode surface and the amperometric responses were measured at -0.20V (vs. Ag pseudo-reference electrode), upon addition of hydroquinone (HQ) as electron transfer mediator and H2O2 as the enzyme substrate. After a thorough optimization of the assay, extremely low limits of detection were achieved: 2.0pg mL(-1) (36fM) and 5.8pg mL(-1) (105fM) for standard solutions and spiked human serum, respectively. The simplicity, robustness and this clinically interesting LOD proved the developed TNFα immunoassay as a good contender for real clinical application.