Cancer-associated extracellular vesicles (EVs) are crucial biomarkers for cancer diagnosis due to their rich content of tumor cell information. To efficiently and accurately detect cancer-associated EVs, an electrochemical hydrogen peroxide reduction reaction (HPRR) based biosensor was developed, utilizing enzyme-linked immunosorbent and diatomic catalyst catalytic HPRR current amplification strategies for specific identification and highly sensitive detection. Anti-CXCR4 antibody was immobilized on Au-plated electrode to selectively capture EVs from the sample. Subsequently, Fe/Cu diatomic catalysts, modified with an anti-CD63 antibody, were bounded to the CD63 on the EVs. Quantitative detection of EVs was achieved by measuring the electrical signals from HPRR catalyzed by the labeled Fe/Cu diatomic catalysts. Under optimized conditions, the electrochemical signals exhibited a linear relationship with EV concentration in the range of 500 to 107 particles mL-1, with a detection limit of 117 particles mL-1, maintaining accuracy even in FBS. With its affordability, high sensitivity, and ease of use, this sensor holds significant potential for medication guidance and postoperative evaluation.

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