Ultrasensitive electrochemical detection of avian influenza A (H7N9) virus DNA based on isothermal exponential amplification coupled with hybridization chain reaction of DNAzyme nanowires
Ultrasensitive electrochemical detection of avian influenza A (H7N9) virus DNA based on isothermal exponential amplification coupled with hybridization chain reaction of DNAzyme nanowires
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DOI：
10.1016/j.bios.2014.09.080
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发表时间：
2015-02-15
影响因子：
12.6
通讯作者：
Yao, Meicun
中科院分区：
文献类型：
--
作者：
Yu, Yanyan;Chen, Zuanguang;Yao, Meicun
本文基于DNA酶纳米线的等温指数扩增（EXPAR）和杂交链反应（HCR），建立了一种简单、无标记、具有双重扩增策略的DNA检测电化学生物传感器。通过合理的设计，使固定在电极表面的双链探针与引物和含分子信标的DNA酶均不发生反应。一旦用靶标攻击，双链体探针切割并触发EXPAR介导的靶标再循环和再生循环以及HCR过程。结果，产生更大量的靶以切割双链体探针。随后，由G-四链体单元组成的纳米线通过与固定在电极表面上的链杂交而自组装。在氯化血红素的存在下，形成催化G-四链体-氯化血红素HRP模拟DNA酶。电化学信号可以通过测量氧化的3.3 '，5.5'-四甲基联苯胺硫酸盐（TMB）的还原电流的增加来获得，所述氧化的3.3'，5.5'-四甲基联苯胺硫酸盐（TMB）由DNAzyme在H2O2存在下产生。该方法对H7N9禽流感病毒DNA序列的检测灵敏度为9.4fM，检测范围为4个数量级。该生物传感器还能够区分伴随的DNA序列之间的单核苷酸差异，并在加标的细胞裂解物中表现良好。(C)© 2014 Elsevier B.V.保留所有权利。
In this work, a simple and label-free electrochemical biosensor with duel amplification strategy was developed for DNA detection based on isothermal exponential amplification (EXPAR) coupled with hybridization chain reaction (HCR) of DNAzymes nanowires. Through rational design, neither the primer nor the DNAzymes containing molecular beacons (MBs) could react with the duplex probe which were fixed on the electrode surface. Once challenged with target, the duplex probe cleaved and triggered the EXPAR mediated target recycle and regeneration circles as well as the HCR process. As a result, a greater amount of targets were generated to cleave the duplex probes. Subsequently, the nanowires consisting of the G-quadruplex units were self-assembled through hybridization with the strand fixed on the electrode surface. In the presence of hemin, the resulting catalytic G-quadruplex-hemin HRP-mimicking DNAzymes were formed. Electrochemical signals can be obtained by measuring the increase in reduction current of oxidized 3.3',5.5'-tetramethylbenzidine sulfate (TMB), which was generated by DNAzyme in the presence of H2O2. This method exhibited ultrahigh sensitivity towards avian influenza A (H7N9) virus DNA sequence with detection limits of 9.4 fM and a detection range of 4 orders of magnitude. The biosensor was also capable of discriminating single-nucleotide difference among concomitant DNA sequences and performed well in spiked cell lysates. (C) 2014 Elsevier B.V. All rights reserved.