Exosomes are secreted in the early stage of cancer at low levels that are difficult to detect due to the complex interferents of the sample matrix. Improved cancer detection requires the development of an accurate, reliable, and reusable method to assay exosomes. Here, a regenerable electrochemical biosensor was developed for exosomes detection based on the dual-recognition proximity binding-induced DNA walker and on-off-on strategy. First, we designed two proximity probes, which composed of a Pb²⁺-dependent DNAzyme tail sequence and identifying units (cholesterol and aptamer). By integrating two proximity probes with target exosomes, a dual-recognition proximity binding-induced DNA walker was achieved to convert one exosome to abundant intermediate DNA strands. Second, the conformational change of hairpin DNA (H) was observed with the hybridization of intermediate DNA, resulting in the increment of the distance between electroactive tag and electrode surface (“off” state of the sensor). These signal changes allowed the quantitative measurement of exosomes. Exonuclease treatment resulted in the hydrolysis of the intermediate DNA and the original hairpin structure, restoring this sensor to the “on” state. With the accurate identification of dual-recognition, the perfect signal amplification of DNA walker and the satisfactory reproducibility of an on-off-on strategy, quantitative detection of exosomes was realized in a wide range from 5.0 × 10⁴ to 1 × 10⁸ particles/mL, with a detection limit of 1.6 × 10⁴ particles/mL. Overall, this approach illustrated a promising potential for exosomes distinguishment of cancer cells from normal cells, even in the complex sample matrix.

外泌体在癌症早期以低水平分泌，由于样品基质的复杂干扰物而难以检测。改进癌症检测需要开发一种准确、可靠且可重复使用的方法来检测外泌体。在这里，基于双识别邻近结合诱导的 DNA walker 和开关策略，开发了一种可再生电化学生物传感器用于外泌体检测。首先，我们设计了两个接近探针，由 Pb ²⁺依赖的 DNAzyme 尾序列和识别单位（胆固醇和适体）。通过将两个邻近探针与目标外泌体整合，实现了双识别邻近结合诱导的 DNA walker，将一个外泌体转化为丰富的中间 DNA 链。其次，随着中间DNA的杂交，观察到发夹DNA（H）的构象变化，导致电活性标签和电极表面之间的距离增加（传感器的“关闭”状态）。这些信号变化允许外泌体的定量测量。核酸外切酶处理导致中间体 DNA 和原始发夹结构的水解，将该传感器恢复到“开启”状态。双重识别精准识别，⁴至 1 × 10 ⁸颗粒/mL，检测限为 1.6 × 10 ⁴颗粒/mL。总体而言，即使在复杂的样品基质中，这种方法也显示了外泌体区分癌细胞与正常细胞的潜力。