Ultrasensitive Profiling of Plasma Extracellular Vesicles for Breast Cancer Subtyping with a High-Curvature Antifouling Nanoarray

Profiling of plasma extracellular vesicles (EVs) has long been hampered by their insufficient capture and assay sensitivity due to the high background of complex matrices. To address this challenge, we develop a high-curvature antifouling nanoarray electrochemical assay (eCAN) to enable ultrasensitive and specific profiling of plasma EVs for the accurate subtyping of breast cancer. This assay leverages a three-in-one multifunctional hierarchical antifouling nanofilm to improve EV capture, minimize nonspecific adsorption, and facilitate three-dimensional deposition of tyramine for signal amplification. These advantages allow the eCAN to achieve a sensitivity of up to 56 particles/mL (near a single-EV level), showing high specificity and anti-interference. The eCAN can differentiate EV subpopulations across different breast cancer cells and monitor their phenotypic changes. This assay allows accurate diagnosis and subtyping of breast cancer (AUC = 1.000) through direct profiling of EVs in undiluted plasma from a pilot cohort and provides a promising tool for precise diagnosis of cancers in clinical settings.