一种新型的基于液滴的自动化芯片PCR系统的研究

How to develop more effective methods for early diagnosis of cancer has become one of the hot research subject of chemistry analysis. Generally, the etiology of cancer is considered as intracellular gene mutation. Therefore, early stage of cancer could be identified by detecting the mutation of gene sequence. Since cancer cell concentration is very low on the early stage, different signal amplification methods are performed in existing detection methods in order to achieve the purpose of diagnosis. It is different for current method for early detection to combine with other methods, and it is also very hard to integrate into an automatic system, so the aim of this project is to develop a new automatic on chip PCR system using droplets. Firstly, the PCR chip system channel surface will be selective modified, so that the positive and negative alternating electroosmotic pump could be build using organic ionic liquids. After the voltage was applied, organic ionic liquids inside the channel will generate electroosmotic flow which will drive the small aqueous droplets contains PCR reaction mixture moving in a closed channel with different temperature area in cycle, and finish the amplification of DNA. The laser induced fluorescence detector is used for quantitative detection on each cycle of amplification. An automatic LabVIEW software is used for controlling the amplification and automatic sampling. This system will promote the development of micro total analysis system (µTAS), and will give a good sample for miniaturization and integration for test and analysis instrument.

如何开发出更有效的癌症早期诊断方法已经成为分析化学学科的研究热点之一。一般认为癌症的病因在于细胞内的基因突变。因此，可以利用检测突变的基因序列来进行癌症早期检测。由于早期阶段各种癌症细胞的含量较低，目前对于现有的检测方法来讲，需要经过各种手段来进行信号的放大从而达到检测目的。针对现有的早期检测方法难于与其他方法联用，集成化和自动化程度低等问题，本项目拟开发一种新型的基于液滴的自动化芯片PCR系统。利用在PCR芯片通道内表面进行选择性修饰，构筑基于有机离子液体的正负交替电渗泵；施加电压后通道内的有机离子液体将会在电渗流的驱动下，带动含有PCR反应液的水相溶液小液滴在一个闭合的通道内不同温度区域间循环移动，完成扩增；并且利用激光诱导荧光检测器定量检测每轮扩增，通过软件自动控制扩增的终止以及自动进样。该系统将推动微全分析系统的发展，也对于分析测试仪器的小型化和集成化发展研究有重要的借鉴意义。

癌症的早期诊断近年来逐渐成为分析化学的热门领域。早期癌症的诊断途径之一是检测癌细胞突变的特异型DNA序列。由于早期阶段癌细胞的个数较少，因此高灵敏度的检测仪器和信号放大策略亟待开发。本项目开发了一种微型化共聚焦式激光诱导荧光芯片电泳仪和建立了基于聚鸟嘌呤四聚体的DNA序列检测信号放大体系。首先设计出共聚焦式激光诱导荧光检测器原型机，并使用原型机利用分子自发荧光检测了异喹啉类生物碱和姜黄素类化合物，结果表明原型机性能良好。在原型机的基础上，通过将光路系统和高压模块，气体控制模块，样品采集模块集成与一体，研制出了尺寸为15 cm（长）X 10 cm（宽）X 20 cm（高）的微型化芯片电泳仪。对于DNA的检测，利用互补序列碱基配对，设计了一种可以进行信号放大的DNA机器，利用生成的聚鸟嘌呤四聚体的催化活性对信号进行进一步放大，从而实现低含量DNA片段的高灵敏检测。