We propose a ratiometric electrochemical assay for detecting microRNA (miRNA) on the basis of dual-amplification mechanism by using distinguishable electrochemical signals from thionine (Thi) and ferrocene (Fc). The thiol-modified and ferrocene-labeled hairpin capture probes (CP) are first immobilized on an Au electrode via Au-S reaction. The target miRNA hybridizes with CP and unfolding the hairpin structure of CP to form miRNA-DNA duplexes. Then, kamchatka crab duplex specific nuclease (DSN) specifically cleaves the DNA in miRNA-DNA duplexes, leading to the release of miRNA and another cleaves cycle, meanwhile, numerous Fc leaves away from the electrode surface and leads to the signal-off of Fc. The residual fragment on electrode surface acts as a HCR primer to form dsDNA polymers through in situ HCR with the presence of the primer and two probes (HDNA and HDNA’), resulting in the capture of numerous DNA/Au NPs/Thi and the signal-on of Thi. The dual-amplification mechanism significantly amplifies the decrease of Fc signal and the increase of Thi signal for ratiometric readout (I_Thi/I_Fc), thus providing a sensitive method for the selective detection of miR-141 with a detection limit down to 11 aM. The dual-signal ratiometric outputs have an intrinsic self-calibration to the effects from system, which is promising to be applied in biosensing and clinical diagnosis.

我们提出了一种基于比例扩增的电化学检测方法，该方法基于双重扩增机制，通过使用可区分的来自硫氨酸（Thi）和二茂铁（Fc）的电化学信号来检测微RNA（miRNA）。首先通过Au-S反应将巯基修饰的和二茂铁标记的发夹捕获探针（CP）固定在Au电极上。目标miRNA与CP杂交并展开CP的发夹结构，形成miRNA-DNA双链体。然后，堪察加蟹双链特异性核酸酶（DSN）特异性切割miRNA-DNA双链体中的DNA，导致miRNA的释放和另一个切割周期，同时，大量Fc离开电极表面并导致Fc的信号关闭。 。电极表面上的残留片段充当HCR引物，在存在引物和两个探针（HDNA和HDNA'）的情况下通过原位HCR形成dsDNA聚合物，从而捕获了许多DNA / Au NP / Thi和信号-Thi。双重放大机制可显着放大Fc信号的减少和Thi信号的增加，以实现比率式读数（I_Thi / I _Fc），因此提供了一种灵敏的方法，可选择性检测miR-141，检测限低至11 aM。双信号比例输出具有对系统影响的内在自校准，有望在生物传感和临床诊断中应用。