Ultrasensitive Electrochemical Detection of Nucleic Acids Based on the Dual-Signaling Electrochemical Ratiometric Method and Exonuclease III-Assisted Target Recycling Amplification Strategy
Ultrasensitive Electrochemical Detection of Nucleic Acids Based on the Dual-Signaling Electrochemical Ratiometric Method and Exonuclease III-Assisted Target Recycling Amplification Strategy
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基于双信号电化学比率法和核酸外切酶III辅助靶标回收放大策略的核酸超灵敏电化学检测
DOI：
10.1021/acs.analchem.5b01402
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发表时间：
2015
影响因子：
7.4
通讯作者：
Chen Jinhua
中科院分区：
文献类型：
--
作者：
Xiong Erhu;Zhang Xiaohua;Liu Yunqing;Zhou Jiawan;Yu Peng;Li Xiaoyu;Chen Jinhua
由于核酸作为生物靶标的内在重要性，核酸的简单和灵敏的检测对于生物学研究和医学诊断是非常必要的。基于双信号电化学比率法和核酸外切酶III（Exo III）辅助的靶循环扩增策略，开发了一种新型、简单、选择性高的电化学DNA生物传感器，用于靶DNA（T-DNA）的灵敏检测。测定策略包括“信号开启”和“信号关闭”元素。以巯基亚甲基蓝（MB）和二茂铁（Fc）为标记物的茎环（发夹）DNA捕获探针（HP）通过Au-S键在金电极表面自组装。在T-DNA存在下，T-DNA与HP杂交，这触发了Exo III切割过程并伴随着T-DNA的释放。结果表明，MB标记远离金电极表面，Fc标记靠近金电极表面，导致MB（IMB）的氧化峰电流降低，Fc（IFc）的氧化峰电流增加。ΔIFc的值/|ΔIMB| T-DNA浓度在0.01 ~ 0.8pM范围内与峰电流呈线性关系（Δ IFC和Δ IMB分别为Fc和MB氧化峰电流的变化值）。所开发的方法的检测限（4.16 fM）是远远低于最报告的电化学DNA生物传感器。该方法为T-DNA的检测提供了一种简单、灵敏的方法，在生物分析、疾病诊断和临床生物医学中具有广阔的应用前景。
Because of the intrinsic importance of nucleic acids as biotargets, the simple and sensitive detection of nucleic acids is very essential for biological studies and medical diagnostics. In this work, a novel, simple, and selective electrochemical DNA biosensor for the sensitive detection of target DNA (T-DNA) has been developed based on the dual-signaling electrochemical ratiometric method and exonuclease III (Exo III)-assisted target recycling amplification strategy. The assay strategy includes both “signal-on” and “signal-off” elements. The stem-loop (hairpin) DNA capture probe (HP), which was labeled by thiolated methylene blue (MB) at the 3′-protruding termini and ferrocene (Fc) in the middle of the loop, first self-assembled on the gold electrode surface via a Au–S bond. In the presence of T-DNA, the T-DNA hybridized with HP, which triggered the Exo III cleavage process and accompanied the release of T-DNA. As a result, the MB tags were away from and the Fc tags close to the gold electrode surface, leading to the decrease of the oxidation peak current of MB (IMB) and the increase of that of Fc (IFc). The value of ΔIFc/|ΔIMB| (ΔIFcand ΔIMBare the change values of the oxidation peak currents of Fc and MB, respectively) is linear with the concentration of T-DNA from 0.01 pM to 0.8 pM. The detection limit (4.16 fM) of the developed method is much lower than that of the most reported electrochemical DNA biosensors. This strategy provides a simple and sensitive approach for the detection of T-DNA and has promising applications in bioanalysis, disease diagnostics, and clinical biomedicine.