Sandwich-Type Electrochemical Paper-Based Immunosensor for Claudin 7 and CD81 Dual Determination on Extracellular Vesicles from Breast Cancer Patients

This study is focused on identifying novel epithelial markers in circulating extracellular vesicles (EVs) through the development of a dual sandwich-type electrochemical paper-based immunosensor for Claudin 7 and CD81 determination, as well as its validation in breast cancer (BC) patients. This immunosensor allows for rapid, sensitive, and label-free detection of these two relevant BC biomarkers. Under optimum conditions, the limit of detection for Claudin 7 was 0.4 pg mL–1, with a wide linear range of 2 to 1000 pg mL–1, while for CD81, the limit of detection was 3 pg mL–1, with a wide linear range of 0.01 to 10 ng mL–1. Finally, we validated Claudin 7 and CD81 determination in EVs from 60 BC patients and 20 healthy volunteers, reporting higher diagnostic accuracy than the one observed with classical diagnostic markers. This analysis provides a low-cost, specific, versatile, and user-friendly strategy as a robust and reliable tool for early BC diagnosis.