Organophosphorus pesticides detection using broad-specific single-stranded DNA based fluorescence polarization aptamer assay.
Organophosphorus pesticides detection using broad-specific single-stranded DNA based fluorescence polarization aptamer assay.
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DOI：
10.1016/j.bios.2013.12.020
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发表时间：
2014-05
影响因子：
12.6
通讯作者：
Cunzheng Zhang;Li Wang;Zhui Tu;Xing Sun;Qing-hua He;Zhaojing Lei;Chongxin Xu;Yuan Liu;
中科院分区：
文献类型：
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作者：
Cunzheng Zhang;Li Wang;Zhui Tu;Xing Sun;Qing-hua He;Zhaojing Lei;Chongxin Xu;Yuan Liu;
开发了一种通过分子信标 (MB) 与重组广泛特异性 DNA 适体竞争性结合来检测有机磷农药的方法，MB 与适体的结合导致荧光信号的激活，可以通过测量荧光信号来进行农药定量。通过指数富集配体系统进化（SELEX）选择的适体经过结构修饰和截短以缩小靶标的结合区域，这表明适体的环对不同的化学识别贡献不同的功能。此后，由两个不同的最小功能结构融合的变体被阐明，具有广泛的特异性和增加的亲和力。进行了进一步的分子对接和分子动力学模拟，以了解 DNA 结构和化学物质之间的分子相互作用。 3D 建模揭示了由 3 个结合位点形成的热点区域，包括氢键和范德华相互作用在内的力似乎在实现和稳定化学物质的结合方面发挥着重要作用。最后，基于工程适配体的有机磷农药检测方法成功应用于实际样品测试中，甲拌磷、丙溴磷、水胺磷和氧化乐果的定量限（LOQ）分别达到19.2、13.4、17.2和23.4 nM（0.005 mg L−1）。
An approach is developed to detect the organophosphorus pesticides via competitive binding to a recombinant broad-specificity DNA aptamer with a molecular beacon (MB), the binding of the MB to the aptamer results in the activation of a fluorescent signal, which can be measured for pesticide quantification. Aptamers selected via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) were structurally modified and truncated to narrow down the binding region of the target, which indicated that loops of the aptamer contributed different functions for different chemical recognition. Thereafter, a variant fused by two different minimum functional structures, was clarified with broad specificity and increased affinity. Further molecular docking and molecular dynamics simulations was conducted to understand the molecular interaction between DNA structure and chemicals. 3D modeling revealed a hot spot area formed by 3 binding sites, forces including hydrogen bonds and van der Waals interactions appear to play a significant role in enabling and stabilizing the binding of chemicals. Finally, an engineered aptamer based approach for the detection of organophosphorus pesticides was successfully applied in a test using a real sample, the limit of quantification (LOQ) for phorate, profenofos, isocarbophos, and omethoate reached 19.2, 13.4, 17.2, and 23.4 nM (0.005 mg L−1), respectively.