Sensitive and selective detection of microRNAs (miRNAs) in cancer cells derived exosomes have attracted rapidly growing interest owing to their potential in diagnostic and prognostic applications. Here, we design a ratiometric electrochemical biosensor based on bipedal DNA walkers for the attomolar detection of exosomal miR-21. In the presence of miR-21, DNA walkers are activated to walk continuously along DNA tracks, resulting in conformational changes as well as considerable increases of the signal ratio produced by target-respond and target-independent reporters. With the signal cascade amplification of DNA walkers, the biosensor exhibits ultrahigh sensitivity with the limit of detection (LOD) down to 67 aM. Furthermore, owing to the background-correcting function of target-independent reporters termed as reference reporters, the biosensor is robust and stable enough to be applied in the detection of exosomal miR-21 extracted from breast cancer cell lines and serums. In addition, because locked nucleic acid (LNA) modified toehold mediate strand displacement reaction (TMSDR) has extraordinary discriminative ability, the biosensor displays excellent selectivity even against the single-base-mismatched target. It is worth mentioning that our sensor is regenerative and stable for at least 5 cycles without diminution in sensitivity. In brief, the high sensitivity, selectivity and reproducibility, together with cheap, make the proposed biosensor a promising approach for exosomal miRNAs detection, in conjunction with early point-of-care testing (POCT) of cancer.

由于癌细胞在外泌体中的微小RNA（miRNA）的灵敏和选择性检测，由于其在诊断和预后应用中的潜力，吸引了快速增长的兴趣。在这里，我们设计了一种基于双足DNA Walker的比例电化学生物传感器，用于外泌体miR-21的摩尔定量检测。在存在miR-21的情况下，DNA Walker被激活以沿着DNA轨道连续行走，从而导致构象变化以及由靶标反应和独立于靶标的报道基因产生的信号比率显着增加。通过DNA Walker的信号级联扩增，该生物传感器显示出超高灵敏度，检测限（LOD）降至67 aM。此外，由于称为目标报告者的与目标无关的报告者的背景校正功能，该生物传感器具有足够的鲁棒性和稳定性，可用于检测从乳腺癌细胞系和血清中提取的外泌体miR-21。此外，由于锁定核酸（LNA）修饰的脚趾介导链置换反应（TMSDR）具有非凡的判别能力，因此即使针对单碱基不匹配的靶标，该生物传感器也显示出出色的选择性。值得一提的是，我们的传感器可再生且稳定至少5个周期，而灵敏度不会降低。简而言之，高灵敏度，选择性和可重复性再加上廉价，使得所提出的生物传感器与癌症的早期即时检测（POCT）结合，成为一种用于外泌体miRNA检测的有前途的方法。