摘要
PCR反应(Polymerase Chain Reaction,聚合酶链式反应)是分子生物学研究不可或缺的手段。传统的PCR技术存在很多缺点,而利用MEMS微加工技术制造出的微流控生物芯片可以在PCR的应用上有着很大的优势。
本文的研究目的是设计并制作出可靠性稳定、成本低、功能集成度高的微流控芯片,并能与外部控制设备相结合,共同组成方便、快捷、高效的PCR扩增检测系统,实现一体化的DNA片段的试样准备、扩增和检测。为进一步的生物应用打下基础。
本文的主要研究内容和成果如下:
1.设计出一套PCR-CE一体化系统,其能一体化的实现试剂的混合、PCR反应、产物分离。微流控芯片主要由具有微通道的上PDMS盖片与溅射了电极的下玻璃基板键合而成。
2.对本文设计的温度和传感电极位于PCR反应腔下的结构进行了有限元热模拟分析。分析证明这种设计拥有更好的均匀热分布,同时温控上也有着突出优势。
3.通过对MEMS工艺的研究,最终确定了一套制作PDMS-玻璃微流控芯片的可靠技术。使用SU-8快速制备阳模,PDMS转移图形得到具有微流控通道的PDMS盖片;在玻璃基板上加工铂电极,然后保护好需要外露部分电极,其他部分以薄层PDMS覆盖,得到电极基板;将PDMS盖片与电极基板半固化键合制得同时具有加热和温度传导电极和CE高压电极的PDMS-玻璃芯片。
4.搭建了PCR-CE微流控检测系统的相关的外部设备和程序,并对微流控系统进行了测试。温控系统升温速度达到15°C / s,波动范围为±0.4°C。CE系统也实现了进样、分离与检测。
Polymerase Chain Reaction is an indispensable research method for Molecular Biology. Tradition PCR technology has some defects, while the Microfluidic chip based on MEMS show great advantage in the usage of PCR.
The paper eager to design and fabricate reliable, inexpensive and high-lever integrated microfluidic chips, which can link with external control device. They make a convenient, high-speed and effective PCR amplification and detection system to prepare, amplify and detect DNA fragment in one chip. That will build the base for further biological development.
The main research content and achievement are as follows:
1. Design a PCR-CE integrative microfluidic system, which can accomplish reagent mixture, PCR amplification and product separation detection. The microfluidic is made up of a PDMS cover plate with micro channel and a glass wafer with sputter-deposited electrodes. Cover plate and wafer bond together and form the chip.
2. In our design chip temperature detecting electrodes is directly place under PCR reaction chamber, separated only by thin PDMS membrane. Thermal simulation prove that structure has a better temperature distribution and predominant in temperature control.
3. The paper confirms a set reliable fabrication process of PDMS-Glass microfluidic chip, based on the research of MEMS technology. Male mould was made by SU-8 and the pattern was transferred into PDMS cover plate; The platinum electrode was sputter-deposited on a glass wafer, covered by PDMS membrane except the area need reservation; After partial curing bonded the PDMS cover and electrode substrate, the microfluidic chip has been made. The microfluidic chip integrates heating electrodes, temperature detecting electrodes and high-voltage CE electrodes.
4. The paper built the external device and program and tested the microfluidic system. The heating speed of PCR reaction chamber is up to more than 15°C / s controlling accuracy is±0.4°C. Capillary Electrophoresis (CE) system also accomplishes the experiment of introduction, separation and detection.
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