Ratiometric electrochemical assay for sensitive detecting microRNA based on dual-amplification mechanism of duplex-specific nuclease and hybridization chain reaction
Ratiometric electrochemical assay for sensitive detecting microRNA based on dual-amplification mechanism of duplex-specific nuclease and hybridization chain reaction
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基于双链体特异性核酸酶和杂交链式反应双扩增机制的比率电化学分析法灵敏检测microRNA
DOI：
10.1016/j.bios.2017.11.030
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发表时间：
2018
影响因子：
12.6
通讯作者：
Qiu Jian-Ding
中科院分区：
文献类型：
--
作者：
Yuan Yan Hong;Chi Bao-Zhu;Wen Shao-Hua;Liang Ru-Ping;Li Zhi-Mei;Qiu Jian-Ding
基于双放大机制，提出了一种基于比例电化学检测microRNA（miRNA）的新方法，该方法利用噻吩（Thi）和二茂铁（Fc）的电化学信号进行区分。首先通过Au-S反应将巯基修饰和二茂铁标记的发夹捕获探针（CP）固定在Au电极上。靶miRNA与CP杂交，解折叠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）特异性地切割miRNA-DNA双链体中的DNA，导致miRNA的释放和另一个切割循环，同时，大量的Fc离开电极表面，导致Fc的信号关闭。电极表面上的残留片段充当HCR引物，在引物和两种探针（HDNA和HDNA '）的存在下通过原位HCR形成dsDNA聚合物，导致大量DNA/Au NP/Thi的捕获和Thi的信号开启。双扩增机制显著放大了Fc信号的降低和Thi信号的增加，用于比率读数（IThi/IFc），从而提供了用于选择性检测miR-141的灵敏方法，检测限低至11 aM。双信号比例输出对系统的影响具有内在的自校准能力，在生物传感和临床诊断中具有广阔的应用前景。
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 (IThi/IFc), 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.