Ultrasensitive fluorescence detection of nucleic acids using exonuclease III-induced cascade two-stage isothermal amplification-mediated zinc (II)-protoporphyrin IX/G-quadruplex supramolecular fluorescent nanotags.
Ultrasensitive fluorescence detection of nucleic acids using exonuclease III-induced cascade two-stage isothermal amplification-mediated zinc (II)-protoporphyrin IX/G-quadruplex supramolecular fluorescent nanotags.
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DOI：
10.1016/j.bios.2014.05.047
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发表时间：
2014-11
影响因子：
12.6
通讯作者：
Qingwang Xue;Yanqin Lv;Yuanfu Zhang;Shuling Xu;Rui Li;Qiaoli Yue;Haibo Li;Lei Wang;X. Gu;Shuqiu Zhang;Jifeng Liu
中科院分区：
文献类型：
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作者：
Qingwang Xue;Yanqin Lv;Yuanfu Zhang;Shuling Xu;Rui Li;Qiaoli Yue;Haibo Li;Lei Wang;X. Gu;Shuqiu Zhang;Jifeng Liu
利用磁性纳米粒子（MNP）和核酸外切酶III（Exo III）诱导的级联两阶段等温扩增技术，建立了一种级联传感系统，用于检测超低浓度的DNA靶分子。一个巧妙设计的捕获发夹探针（CHP），整合目标结合和信号转导序列在一个多功能的设计组装在MNP。当检测到分析物核酸时，MNP上的发夹探针可以被打开并被Exo III逐步去除，伴随着靶DNA的释放用于连续的杂交和切割过程，并产生CHP的裸信号转导序列作为下一个循环反应的新触发器。新的DNA触发剂启动与含有部分“笼状”G-四链体序列（GHP）的发夹DNA探针杂交，形成双链体结构并释放活性G-四链体结构。然后，Exo III使所得的双链体结构域发生变性，导致新的DNA触发物的再循环，并同时在锌（II）-原卟啉IX（ZnPPIX）的帮助下产生许多ZnPPIX/G-四链体超分子复合物，作为放大的荧光感测事件的光学标记。最后，许多解放级联ZnPPIX/G-四链体超分子复合物给出了显着的荧光响应。由于两阶段自催化循环扩增和ZnPPIX/G-quadruplex超分子复合物的特异性催化形成，该新设计的方案提供了高灵敏度，检测限为0.75 fM，可以区分错配DNA和完全匹配的靶DNA，并且由于使用MNP作为分离和扩增元件，在真实的样品中具有低基质效应。因此，它在基因相关疾病的早期诊断方面具有很大的潜力。
A cascadic sensing system was developed for detection of DNA target at ultralow concentration by a combination of magnetic nanoparticles (MNPs) and exonuclease III (Exo III)-induced cascade two-stage isothermal amplification in the study. An ingeniously designed capture hairpin probe (CHP) that integrates target-binding and signal transduction sequences within one multifunctional design was assembled on MNPs. Upon sensing of the analyte nucleic acid, the hairpin probe on MNPs could be opened and stepwise removed by Exo III accompanied by the releasing of target DNA for the successive hybridization and cleavage process and the generation of bare signal transduction sequences of CHP as a new trigger for next circular reaction. The new DNA triggers initiate hybridizing with hairpin DNA probe that contains a partially “caged” G-quadruplex sequence (GHP), forming a duplex structure and liberating the active G-quadruplex structure. Then, Exo III digests the resulting duplex domain, leading to the recycling of new DNA trigger and simultaneously generating numerous ZnPPIX/G-quadruplex supramolecular complexes with the help of the zinc (II)-protoporphyrin IX (ZnPPIX), as an optical label for amplified fluorescence sensing event. Finally, numerous liberated cascade ZnPPIX/G-quadruplex supramolecular complexes give a remarkable fluorescence response. Because of two-stage autocatalytic recycling amplification and the specifically catalyzed formation of ZnPPIX/G-quadruplex supramolecular complexes, this newly designed protocol provides a high sensitivity with a detection limit of 0.75 fM, can discriminate mismatched DNA from perfectly matched target DNA, and gives low matrix effect due to using MNPs as the separation and amplification elements in the real samples. Therefore, it holds great potential for early diagnosis in gene-related diseases.