Mei Qiang, Gu Baiwen, Jiang Yinyu, Wang Yulin, Lai Weiju, Chen Hu, Chen Jide, Zhao Xianxian
Equipment Trading Division, Chongqing Pharmaceutical Exchange Co., Ltd., Chongqing 401336, China.
Central Laboratory, Chongqing University FuLing Hospital, Chongqing 408099, China.
ACS Omega. 2024 Mar 21;9(13):15350-15356. doi: 10.1021/acsomega.3c09888. eCollection 2024 Apr 2.
Signal amplification strategies have emerged as a prominent tool in the field of improving the detection sensitivity of small extracellular vesicles (sEVs). It is important to highlight that the utilization of signal quenching strategies is not commonly implemented. A detection technique for sEVs was established based on the unwinding of G-quadruplex using Klenow fragment polymerase (KF), which served as an inspiration for this study. This system is characterized by its simplicity and lack of labeling, making it an efficient approach for signal quenching. In the presence of sEVs, the CD63 aptamer in the capture@sMBs complex binds with the CD63 protein on the surface of sEVs to release trigger sequences, which were employed as a primer to mediate the DNA polymerase/endonuclease-assisted signal recycling. The signal recycling process produces numerous single-stranded DNA sequences that can bind to the toehold section of the G-quadruplex. This leads to the rupture of the G-quadruplex structure and the subsequent deactivation of a DNAzyme generated by the G-quadruplex structure and hemin, thereby inhibiting its biological catalytic function. Consequently, the G-quadruplex structure would undergo a transformation to a duplex structure, leading to the emergence of a discernible differential signal that can be noticed in a majority of instances, even without the aid of magnification devices. The decrease in the prominent signal allows for the efficient analysis of target sEVs, which exhibit a notably low detection limit. In addition to the detection of sEVs, the approach has also been utilized for the investigation of miRNA-21. The approach demonstrates a high level of selectivity and robustness in its capacity to differentiate between target miRNA and base-mismatched miRNA as well as other miRNA families. This statement suggests that the assay holds significant promise for use in biochemical research and clinical diagnosis.
信号放大策略已成为提高小细胞外囊泡（sEVs）检测灵敏度领域的一种重要工具。需要强调的是，信号淬灭策略的应用并不常见。基于使用Klenow片段聚合酶（KF）解开G-四链体建立了一种sEVs检测技术，这为本研究提供了灵感。该系统的特点是简单且无需标记，使其成为一种有效的信号淬灭方法。在存在sEVs的情况下，捕获@sMBs复合物中的CD63适体与sEVs表面的CD63蛋白结合以释放触发序列，该序列用作引物介导DNA聚合酶/核酸内切酶辅助的信号循环。信号循环过程产生大量可与G-四链体的起始区域结合的单链DNA序列。这导致G-四链体结构破裂，随后由G-四链体结构和血红素产生的DNAzyme失活，从而抑制其生物催化功能。因此，G-四链体结构将转变为双链结构，导致出现可在大多数情况下被注意到的明显差异信号，甚至无需借助放大装置。显著信号的降低使得能够对目标sEVs进行有效分析，其检测限极低。除了检测sEVs外，该方法还被用于研究miRNA-21。该方法在区分目标miRNA与碱基错配的miRNA以及其他miRNA家族的能力方面表现出高度的选择性和稳健性。这表明该检测方法在生化研究和临床诊断中具有巨大的应用前景。
