Assays for cancer diagnosis via the analysis of biomarkers on circulating extracellular vesicles (EVs) typically have lengthy sample workups, limited throughput or insufficient sensitivity, or do not use clinically validated biomarkers. Here we report the development and performance of a 96-well assay that integrates the enrichment of EVs by antibody-coated magnetic beads and the electrochemical detection, in less than one hour of total assay time, of EV-bound proteins after enzymatic amplification. By using the assay with a combination of antibodies for clinically relevant tumour biomarkers (EGFR, EpCAM, CD24 and GPA33) of colorectal cancer (CRC), we classified plasma samples from 102 patients with CRC and 40 non-CRC controls with accuracies of more than 96%, prospectively assessed a cohort of 90 patients, for whom the burden of tumour EVs was predictive of five-year disease-free survival, and longitudinally analysed plasma from 11 patients, for whom the EV burden declined after surgery and increased on relapse. Rapid assays for the detection of combinations of tumour biomarkers in plasma EVs may aid cancer detection and patient monitoring.

通过分析循环细胞外囊泡 (EV) 上的生物标志物进行癌症诊断的检测通常需要冗长的样本处理、有限的通量或灵敏度不足，或者不使用经过临床验证的生物标志物。在这里，我们报告了一种 96 孔检测的开发和性能，该检测结合了抗体包被磁珠对 EV 的富集和酶促扩增后 EV 结合蛋白的电化学检测，总检测时间不到一小时。通过结合使用结直肠癌 (CRC) 临床相关肿瘤生物标志物（EGFR、EpCAM、CD24 和 GPA33）的抗体，我们对来自 102 名 CRC 患者和 40 名非 CRC 对照者的血浆样本进行了分类，准确度超过96%，前瞻性地评估了一组 90 名患者，对他们而言，肿瘤 EV 的负担可预测五年无病生存率，并纵向分析了 11 名患者的血浆，对于这些患者，EV 负担在手术后下降并在复发时增加。用于检测血浆 EV 中肿瘤生物标志物组合的快速检测可能有助于癌症检测和患者监测。