基于MEMS工艺的PCR微流控系统的研制
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.
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.
引文
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[33] Y. Schaerli, R.C. Wootton, T. Robinson et al. Continuous-Flow Polymerase Chain Reaction of Single-Copy DNA in Microfluidic Microdroplets. Anal. Chem. 81 (2009): 302-306
[34] Isabel Rodriguez, Marie Lesaicherre, Yan Tie et al. Practical integration of polymerase chain reaction amplification and electrophoretic analysis in microfluidic devices for genetic analysis. ELECTROPHORESIS, 24: 172–178
[35] E. T. Lagally, J. R. Scherer, R. G. Blazej et al. Mathies. Integrated Portable Genetic Analysis Microsystem for Pathogen/Infectious Disease Detection. Anal. Chem., 2004, 76 (11): 3162–3170
[36] Chung N. Liu, Nicholas M. Toriello, and Richard A. Mathies. Multichannel PCR-CE Microdevice for Genetic Analysis. Analytical Chemistry 2006 78 (15): 5474-5479
[37] Xiaoyan Pan, Lei Jiang, Kaiying Liu et al. A microfluidic device integrated with multichamber polymerase chain reaction and multichannel separation for genetic analysis, Analytica Chimica Acta, Volume 674, Issue 1, 26 July 2010, 110-115
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[40]贾晓宇.集成式连续流PCR芯片及制造技术的研究[学位论文]。上海交通大学,2006.
[41]陈实.用于微流体芯片的PDMS被动式微混合器研究[学位论文]。上海交通大学,2007.
[42] Niu Z Q, Chen W Y, Shao S Y, et al. DNA amplification on a PDMS-glass hybrid microchip [J]. Journal of Micromechanics and Microengineering. 2006, 16(2): 425-433.
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[02]方肇伦,程介克,陈洪渊,常文宝,邹汉法,微流控分析芯片[M],科学出版社, (2003),北京
[03]章吉良,杨春生等,微机电系统及其相关技术[M],上海:上海交通大学出版社,2000.1
[04]罗均,谢少荣,面向MEMS的微细加工技术[M],电加工和模具,2001.5,1-6
[05]苑伟政,马炳和.微机械与微细加工技术[M],西安:西北工业大学出版社, 2001.
[06] Koutny L B, Schmalzing D, Taylor T A, Fuchs M. Microchip Electrophoretic Immunoassay for Serum Cortisol. Anal. Chem, 1996. 68: 18.22.
[07] Kwang W Oh and Chong H Ahn. A review of microvalves. Journal of Micromechanics and Microengineering 2006.16: 13-39
[08]苏波,崔大付,刘长春等。微流控光纤芯片检测系统的研制。分析化学,2006,34:295-298.
[09] Michael L. Chabinyc, Daniel T. Chiu, J. Cooper McDonald et al. An Integrated Fluorescence Detection System in Poly(dimethylsiloxane) for Microfluidic Applications. Analytical Chemistry 2001 73 (18), 4491-4498
[10] Shize Qi, Xuezhu Liu, Sean Ford et al. Microfluidic devices fabricated in poly(methylmethacrylate) using hot-embossing with integrated sampling capillary and fiber optics for fluorescence detection. Lab Chip, 2002, 2, 88-95
[11] Hai F L, Jin M L, Rong G S et al. A compactly integrated laser-induced fluorescence detector for microchip. Electrophoresis, 2004, 25: 1907-1915.
[12]许成才.光纤嵌入式毛细管电泳芯片的信号检测及其小波消噪[学位论文].大连理工大学,2006.
[13]林炳承,秦建华.微流控芯片实验室[M].科学出版社.2006:123～137
[14] M.A. Northrup, M.T. Ching, R.M. White and R.T. Watson, DNA amplification in a microfabricated reaction chamber Proc. 7th International Conference on Solid-State Sensors and Actuators (Transducers '93)Yokohama, Japan (7–10 June 1993), pp. 924–926.
[15] S. Poser, T. Schulz, U. Dillner et al. Chip Elements for Fast Thermocycling. Sensors and Actuators A. 1997, 62(1-3): 672~675.
[16] Quanbo, Zoua, Yubo, Miaoa, Yu, Chena, Micro-assembled multi-chamber thermal cycler for low-cost reaction chip thermal multiplexing, Sensors and Actuators A, (2002) 102, 114–121
[17] Cheng J, Shoffner MA, Hvichia GE et al. Investigation of different PCR amplification systems in microfabricated silicon glass chips. Nucleic Acids Res, (1996), 24, 380-385
[18] Young, S. S., Keunchang, C., Sun, H., PDMS-based micro PCR chip with Parylene coating, J. Micromech. Microeng, (2003), 13, 768–774
[19] Jung-Hao Wang, Liang-Ju Chien, Tsung-Min Hsieh et al. A miniaturized quantitative polymerase chain reaction system for DNA amplification and detection, Sensors and Actuators B: Chemical, 2009,Volume 141, Issue 1, 329-337.
[20] M.U. Kopp, A.J. de Mello, A. Manz. Chemical amplification: continuous-flow PCR on a chip. Science 280 (1998): 1046-1048
[21] Schneega?, R. Br?utigam, J.M. K?hler. Miniaturized flow-through PCR with different template types in a silicon chip thermocycler. Lab Chip 1 (2001): 42-49
[22] K. Sun, A. Yamaguchi, Y. Ishida et al. A heater-integrated transparent microchannel chip for continuous-flow PCR. Actuators B Chem. 84 (2002): 283-289
[23] P.J. Obeid, T.K. Christopoulos. Continuous-flow DNA and RNA amplification chip combined with laser-induced fluorescence detection. Anal. Chim. Acta 494 (2003): 1-9
[24] P.J.Obeid, T.K. Christopoulos, H.J. Crabtree, C.J. Backhouse. Microfabricated Device for DNA and RNA Amplification by Continuous-Flow Polymerase Chain Reaction and Reverse Transcription-Polymerase Chain Reaction with Cycle Number Selection. Anal. Chem. 75 (2003) 288–295
[25] N Crews, CT Wittwer, J Montgomery. Spatial DNA Melting Analysis for Genotyping and Variant Scanning. Anal. Chem., 2009, 81 (6), pp 2053–205
[26] J. West, B. Karamata, B. Lillis, J.P. Gleeson et al. Application of magnetohydrodynamic actuation to continuous flow chemistry. Lab Chip 2 (2002) 224-230
[27] Jian Liu, Markus Enzelberger, S. Quake. A nanoliter rotary device for polymerase chain reaction, Electrophoresis, (2002), 23, 1531-1536
[28] JiaXiaoyu, NiuZhiqiang, Zhangweiping, Chenwenyuan, Polydimethylsiloxane,(PDMS)-based Spiral Channel PCR chip. IEE Electronics Letters, 43,690-691(SCI)
[29] S. Mohr, Y.-H. Zhang, A. Macaskill et al. Numerical and experimental study of a droplet-based PCR chip. Microfluidics and Nanofluidics, 2007, Volume 3, Number 5, Pages 611-621
[30] N.R. Beer, B.J. Hindson, E.K. Wheeler et al. On-Chip, Real-Time, Single-Copy Polymerase Chain Reaction in Picoliter Droplets. Anal. Chem. 79 (2007): 8471–8475
[31] N.R. Beer, E.K. Wheeler, L. Lee-Houghton et al. On-Chip Single-Copy Real-Time Reverse-Transcription PCR in Isolated Picoliter Droplets. Anal. Chem. 80 (2008): 1854–1858
[32] M.M. Kiss, L. Ortoleva-Donnelly, N.R. Beer et.al. High-Throughput Quantitative Polymerase Chain Reaction in Picoliter Droplets. Anal. Chem. 80 (2008): 8975–8981
[33] Y. Schaerli, R.C. Wootton, T. Robinson et al. Continuous-Flow Polymerase Chain Reaction of Single-Copy DNA in Microfluidic Microdroplets. Anal. Chem. 81 (2009): 302-306
[34] Isabel Rodriguez, Marie Lesaicherre, Yan Tie et al. Practical integration of polymerase chain reaction amplification and electrophoretic analysis in microfluidic devices for genetic analysis. ELECTROPHORESIS, 24: 172–178
[35] E. T. Lagally, J. R. Scherer, R. G. Blazej et al. Mathies. Integrated Portable Genetic Analysis Microsystem for Pathogen/Infectious Disease Detection. Anal. Chem., 2004, 76 (11): 3162–3170
[36] Chung N. Liu, Nicholas M. Toriello, and Richard A. Mathies. Multichannel PCR-CE Microdevice for Genetic Analysis. Analytical Chemistry 2006 78 (15): 5474-5479
[37] Xiaoyan Pan, Lei Jiang, Kaiying Liu et al. A microfluidic device integrated with multichamber polymerase chain reaction and multichannel separation for genetic analysis, Analytica Chimica Acta, Volume 674, Issue 1, 26 July 2010, 110-115
[38]方肇伦,殷学锋,方群等.微流控分析芯片[M]。北京:科学出版社,2003.
[39]牛志强.集成微流PCR聚合物芯片的研究[学位论文]。上海交通大学,2006.
[40]贾晓宇.集成式连续流PCR芯片及制造技术的研究[学位论文]。上海交通大学,2006.
[41]陈实.用于微流体芯片的PDMS被动式微混合器研究[学位论文]。上海交通大学,2007.
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