环介导等温扩增H+诱导DNA纳米结构构型转换超灵敏HIV标志物DNA电化学生物传感器研究

As an emerging nucleic acid amplification technology, loop-mediated isothermal amplification (LAMP) is identified by using visualization methods such as fluorescence, colorimetry and gel electrophoresis. The detection of LAMP reaction byproducts including pyrophosphate and hydrogen ion (H+) could be carried out with turbidimetry and pH meter. However, these methods are of low sensitivity, resulting in difficult ultrasensitive quantitative measurement of target DNA for LAMP. Therefore, based on the target transduction induced by the byproducts (such as H+), which are produced in LAMP reaction triggered by the targeted DNA, it is very significant to develop novel analytical methods with ultra sensitivity (such as electrochemical biosensors). In the present project, human immunodeficiency virus (HIV) biomarker DNA is used as tested model for LAMP. The generated byproduct H+ in LAMP can induce the conformational switch of pH-responsive DNA nanostructures to form stable triplex structures, accompanying with the releasing of specific DNA sequences. The transduction and amplification of the output signals for the released DNA can be achieved by integrating with different biological amplification technologies involving in chain displacement, catalytic hairpin self-assembly, enzymatic cleavage, and etc.. Novel ultrasensitive electrochemical biosensors are developed for HIV biomarkers DNA based on the proposed strategies, which could provide new methods to extend the target DNAs capable of being used in LAMP reaction (such as hepatitis B virus, pathogenic avian influenza virus, and etc.), and promote the development of biosensors and clinical medical examination.

新兴核酸放大技术环介导等温扩增（LAMP）的鉴定主要基于荧光、比色或凝胶等可视化方法，而LAMP反应副产物焦磷酸和H+的检测可利用沉淀浊度法和pH计。但是，这些方法灵敏度低，难以实现扩增目标DNA的超灵敏定量检测。因此，通过目标DNA进行LAMP反应，利用副产物（如H+）进行目标物转换，研制超灵敏新型分析方法（如电化学生物传感器）具有重要意义。本项目以人免疫缺陷病毒（HIV）标志物DNA为分析模型进行LAMP扩增，利用反应副产物H+（pH）诱导具有pH响应的DNA纳米结构构型转换，形成稳定三级结构时释放特异性DNA序列；结合链置换、催化发夹自组装、酶剪切等生物放大技术，对释放的DNA序列进行输出信号转换与放大，构建新型超灵敏HIV标志物DNA电化学生物传感器，为拓展LAMP扩增目标物DNA超灵敏检测范围（或乙肝病毒、高致病禽流感病毒等）、促进生物传感器发展和临床医学检验提供新的研究方法。

环介导等温扩增（LAMP）用于核酸放大具有显著优势。其常规鉴定常用荧光、比色或凝胶等可视化方法，沉淀浊度法和pH计可分别用于副产物焦磷酸和H+（LAMP-H+）的快速检测。但这些方法灵敏度低，难以对目标DNA进行超灵敏定量分析；而传统LAMP的模板链太长，设计较繁琐，且LAMP反应体系复杂多元，易引起明显的背景干扰，这些不足都极大限制了LAMP技术的实际应用。因此，本项目分别以长链DNA标志物（包括家蚕微孢子虫病毒基因272-nt和流感病毒174-nt）直接作为模板进行LAMP扩增，或以人类免疫缺陷病毒标志物短链DNA（21-nt）为模型，通过预增长原位形成双哑铃起始结构，或直接作为外引物，启动LAMP扩增。以LAMP-H+为外部化学刺激转换目标物输入，诱导具有pH依赖性响应的DNA三链体或i-motif二级结构形成等模式，释放序列特异性的DNA探针；在修饰电极传感界面结合链置换、酶剪切、催化发夹自组装、π-π堆积作用等生物放大方法，产生显著的单一或比率型电流输出信号；同时，利用磁性分离，有效降低了复杂多元的LAMP体系引起的假阳性背景干扰；定向结合作用还能极大减小非特异性吸附对电极表面电子转移过程产生的不利影响。本项目中，我们创新性改变LAMP反应模式，以LAMP-H+定向转换目标物输入，通过引入多种放大模式，简化电极反应过程，并与电流响应信号输出一体化结合，成功构建了具有超灵敏、高特异性、快速简便等分析性能的新型DNA电化学生物传感器。项目取得了较好的研究成果，为进一步拓展LAMP技术在生物传感、生物分析等领域的应用提供新的研究范式。

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