Ultrasensitive electrochemical dipstick lateral flow immunoassay for extracellular vesicle quantification using starch-iodine functionalized gold nanoparticles.

Extracellular vesicles (EVs) have emerged as promising biomarkers for the diagnosis and monitoring of various diseases; however, their clinical application is limited by the need for sensitive and accessible detection platforms. In this study, a highly sensitive electrochemical lateral flow immunoassay (eLFIA) for the detection and accurate quantification of EVs in human plasma is presented. Using a polymer precipitation-based kit, EVs were isolated and determined by using iodine-doped starch-functionalized gold nanoparticle based immunochromatographic test. This strategy has never been used before for electrochemical coupling and it has been protected by an international patent. With this electrochemical approach, the biosensor achieved a detection limit of 9 EVs/µL, demonstrating significantly higher sensitivity than conventional optical methods and even exceeding the limits reported by more complex bulk widely-used techniques, such as Nanoparticle Tracking Analysis. In addition, validation on real biological samples confirmed the accuracy of the device with recoveries above 98%, underlining its robustness and reproducibility. This work establishes a versatile and efficient platform for EV detection that addresses key limitations of current immunoassays, positioning this eLFIA as a promising tool for clinical diagnostics. Furthermore, the proposed strategy has the potential to be adapted to other biomarkers, broadening its applicability to a variety of pathologies.