基于MEMS的PCR芯片系统设计研究
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摘要
生物芯片是20世纪具有划时代意义的微量分析检测技术,广泛应用于疾病诊断和治疗、药物筛选、基因测序、中药物种鉴定、农作物优育优选等许多领域。作为生物芯片技术的一种,聚合酶链式反应技术自诞生以来不断发展成熟起来,应用前景越来越广泛。
聚合酶链式反应(Polymerase Chain Reaction, PCR)是一种能够在数小时内使特定DNA片段扩增到数百万个拷贝的体外快速扩增DNA的方法,已广泛应用于生物科学、医学等领域。DNA的扩增过程可分为变性(93~94℃)、退火(54℃)、延伸(72℃)三个连续的、循环的温度操作过程。因此,温度是影响PCR扩增的一个关键因素。
本文设计了基于MEMS技术的PCR芯片系统,在玻璃基片上设计了微反应腔式PCR芯片。芯片上集成了DNA微反应室、加热器及温度传感器,芯片尺寸大小为10mm*10mm*0.5mm,微反应室的尺寸大小为5mm*5mm*0.15mm。芯片底面通过光刻、溅射等工艺制作了微型加热器与温度传感器,以使PCR芯片测温、控温更简便。为使芯片温区温度分布更均匀,通过ANSYS软件对不同结构的加热器进行温度场仿真,得到最优的加热器结构。
PCR芯片温度控制系统的设计,包括硬件和软件两部分。硬件的设计主要包括信号采信、信号调理、CPU系统和控制信号输出几部分。在信号采集部分为避免铂电阻温度传感器因电流流过而产生焦耳热,系统采用0.5mA恒流源电路。采集电路将系统的温度信号转化为电压信号后,经放大、滤波后,由SPCE061A的I/O口采集。采集到的电压信号经计算转化为的温度值后与设定的温度值进行比较,经PID算法计算及相应的比例换算获得相应占空比的PWM方波以控制固态继电器的通断,实现芯片PCR反应温度的自动控制。本文还从理论上证明了PCR芯片系统设计的合理性,并进行了简单的实验来验证系统设计的可行性。
Biochip is a landmark trace analytical techniques developed at the end of last century, which are widely used in disease diagnosis and treatment, drug genome atlas, drug screening, gene sequencing, crop dabbling optimization, food hygiene supervision and many other areas. As a kind of biological chip, PCR chip technology develops ceaselessly, which is a kind of DNA vitro expansion method.
The Polymerase Chain Reaction (PCR) which can make specific DNA segments swell to millions of copies in a matter of hours has been widely applied in biology science, medicine and so on. DNA amplification process can be divided into degeneration (93 ~ 94℃), annealing (54℃), extension (72℃) three consecutive and circulates temperature operation process. Therefore, the temperature is one of the key factors of PCR chip.
The paper designed a micro reaction chamber PCR chip based on MEMS technology in glass. The chip contained a DNA micro-reactor room, a heater and a temperature sensor, which size is about 10mm *10mm* 0.5 mm. Using wet etching methods in glass to etch out a 5mm *5mm * 0.15 mm micro-reactor room. On the other side of the PCR chip there is heater and temperature sensor in order to make the PCR chip miniaturization and integration. To make temperature more uniform, we used ANSYS software to simulate different structures of the heater on temperature field to search for optimal heater structure.
The paper designed a cyclic temperature control system, which included both hardware and software. The hardware design included signal acquisition part, signal disposal part and control signal output part. To avoid platinum generate heating because of large current, signal acquisition circuit adopted 0.5 mA constant-current source circuit. Acquisition circuit transformed temperature signal into voltage signal, which is acquitted by SPCE061A I/O ports after enlargement and filtering. The collected voltage signal is transformed into the value of temperature .After comparing with the setting value and PID calculation, it got a PWM square-wave to control the solid state relay, which realized automatic temperature control.
聚合酶链式反应(Polymerase Chain Reaction, PCR)是一种能够在数小时内使特定DNA片段扩增到数百万个拷贝的体外快速扩增DNA的方法,已广泛应用于生物科学、医学等领域。DNA的扩增过程可分为变性(93~94℃)、退火(54℃)、延伸(72℃)三个连续的、循环的温度操作过程。因此,温度是影响PCR扩增的一个关键因素。
本文设计了基于MEMS技术的PCR芯片系统,在玻璃基片上设计了微反应腔式PCR芯片。芯片上集成了DNA微反应室、加热器及温度传感器,芯片尺寸大小为10mm*10mm*0.5mm,微反应室的尺寸大小为5mm*5mm*0.15mm。芯片底面通过光刻、溅射等工艺制作了微型加热器与温度传感器,以使PCR芯片测温、控温更简便。为使芯片温区温度分布更均匀,通过ANSYS软件对不同结构的加热器进行温度场仿真,得到最优的加热器结构。
PCR芯片温度控制系统的设计,包括硬件和软件两部分。硬件的设计主要包括信号采信、信号调理、CPU系统和控制信号输出几部分。在信号采集部分为避免铂电阻温度传感器因电流流过而产生焦耳热,系统采用0.5mA恒流源电路。采集电路将系统的温度信号转化为电压信号后,经放大、滤波后,由SPCE061A的I/O口采集。采集到的电压信号经计算转化为的温度值后与设定的温度值进行比较,经PID算法计算及相应的比例换算获得相应占空比的PWM方波以控制固态继电器的通断,实现芯片PCR反应温度的自动控制。本文还从理论上证明了PCR芯片系统设计的合理性,并进行了简单的实验来验证系统设计的可行性。
Biochip is a landmark trace analytical techniques developed at the end of last century, which are widely used in disease diagnosis and treatment, drug genome atlas, drug screening, gene sequencing, crop dabbling optimization, food hygiene supervision and many other areas. As a kind of biological chip, PCR chip technology develops ceaselessly, which is a kind of DNA vitro expansion method.
The Polymerase Chain Reaction (PCR) which can make specific DNA segments swell to millions of copies in a matter of hours has been widely applied in biology science, medicine and so on. DNA amplification process can be divided into degeneration (93 ~ 94℃), annealing (54℃), extension (72℃) three consecutive and circulates temperature operation process. Therefore, the temperature is one of the key factors of PCR chip.
The paper designed a micro reaction chamber PCR chip based on MEMS technology in glass. The chip contained a DNA micro-reactor room, a heater and a temperature sensor, which size is about 10mm *10mm* 0.5 mm. Using wet etching methods in glass to etch out a 5mm *5mm * 0.15 mm micro-reactor room. On the other side of the PCR chip there is heater and temperature sensor in order to make the PCR chip miniaturization and integration. To make temperature more uniform, we used ANSYS software to simulate different structures of the heater on temperature field to search for optimal heater structure.
The paper designed a cyclic temperature control system, which included both hardware and software. The hardware design included signal acquisition part, signal disposal part and control signal output part. To avoid platinum generate heating because of large current, signal acquisition circuit adopted 0.5 mA constant-current source circuit. Acquisition circuit transformed temperature signal into voltage signal, which is acquitted by SPCE061A I/O ports after enlargement and filtering. The collected voltage signal is transformed into the value of temperature .After comparing with the setting value and PID calculation, it got a PWM square-wave to control the solid state relay, which realized automatic temperature control.
引文
[1]谢文章,卢英,邢婉丽等.生物芯片——二十一世纪革命性的技术.基础医学与临床,2000 (4):1-4
[2]马立人,蒋中华.生物芯片.第二版.北京:化学工业出版社,2002
[3]周冬生.生物芯片分类及其技术原理.微生物学免疫学进展,2002 (3):101～107
[4]章春笋,徐进良.连续流动式PCR芯片相关技术研究进展.分析测试学报,2004, 23 (6) :114-118
[5]王廷华,景强.PCR理论与技术.北京:科学出版社,2005
[6] Rabinow, P.Making. PCR: A story of biotechnology. Chicago: University of Chicago Press,1996
[7] Kary B.Mullis .The Unusual Origin of the Polymerase Chain Reaction. Scientific American, 1990,262(4):56-61, 64-5
[8] Mullis,K. , Faloona,F. , Scharf,S. , et al. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symposium in Quantitative Biology ,1986, 51: 263-73
[9] Mullis,K. , Faloona,F. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol ,1987,155: 335-350
[10]周天,邹志清,赵建龙,等. PCR微池芯片的设计、制作及实现样品扩增.功能材料与器件学报,2003, 2 (9):191-194
[11]何文波.基于MEMS技术的PCR生物芯片的研究[硕士学位论文].大连:大连理工大学,2003
[12] A.Rolfs, I.Schuller, U.Finck, I.Weber-Rolfs. PCR: Clinical Diagnostics and Research. New York: Springer-Verlag, 1992
[13] M.J.McPhearson, P.Quirke, and G.R.Taylor. PCR: A Practical Approach. Oxford ,UK: Oxford Univ. Press, 1992
[14] K.B.Mullis, F.Ferte, and R.A.Gibbs.The Polymerase Chain Reaction.Boston: Birkhauser, 1994
[15] E.A.Erlich. PCR Technology: Principles and Applications for DNA Amplification. Oxford,UK: Oxford Univ. Press, 1992
[16] P Wilding, Man A.Shoffner and Larry J.Kricka. PCR in a silicon microstructure.Clin Chem, 1994,40:1815-1818
[17] Zhan Zhao, Zheng Cui, Dafu Cui ,et al. Monolithically integrated PCR biochip for DNA amplification. Sensors and Actuators A, 2003, 108:162-167
[18] Martin U. Kopp, Andrew J.deMello, Andreas Manz ,et al .Chemieal Amplification: Continuous-Flow PCR on a Chip . Science,1998,280(15) : 1046-1048
[19]赵湛,崔大付,于中尧,等.基于MEMS技术的PCR生物芯片的研究.传感器技术,2001,20(6):56-59
[20]崔大付.基于BioMEMS技术的DNA芯片研究.机械强度,2001,23(4):471-475
[21]季旭,刘理大.一种集成PCR反应室与CE的生物芯片的研究.仪器仪表学报,2003,24(4):201-202
[22] Isabel Rodrigue Z, Mariel Lesaieherre. Practical integration of polymerase chain reaction amplification and electrophoretic analysis in microfluidic devices fo genetic analysis. Electrophoresis,2003,24:172-178
[23] Toriello NM, Liu CN, Mathies RA. Mutichannel Reverse Transcription-Polymerase Chain Reaction Microdevice for Rapid Gene Expression and Biomarker Analysis. Analytical Chemistry, 2006, 78 (23):7997-8003
[24] Peng Liu,Tae Seok Seo. Integrated Portable Polymerase Chain Reaction-Capillary Electrophoresis Microsystem for Rapid Forensic Short Tandem Repeat Typing. Analytical Chemistry, 2007, 79 (5):1881-1889
[25] Suzanne Cheng, Russell Higuchi , Mark Stoneking. Complete mitochondrial genome amplication. Nature Genet , 1994 , 7 : 350～351
[26] Nelson W S , Prodohl P A , Avise J C. Development and application of long-PCR for the assay of full-length animal mitochondrial DNA. Mol Ecol , 1996 , 5 : 807～810
[27] Ui Wook Hwang , Chan Jong Park , Tai Soon Yong , et al .One-step PCR amplification of complete arthropod mitochondrial genomes. Molecular Phylogenetics and Evolution , 2001 , 19(3) : 345～352
[28] Iseno K.A novel quantitative PCR with fluorogenic probe.Rinsho Byofi,1997,45(3):218-225
[29] Christin Betz, T.Greg McCollum, Richard T.Mayer.Differential expression of two cinnamate 4-hydroxylase genes in’Valencia’orange(Citrus Sinensis Osbeck).PlantMo1ecular Biology,2001,46(6):741-748
[30] J.Bryzek, K.Petersen, W.McCulley. Micromachines on the March. IEEE Spectrum,1994,31(5),20-31
[31] K.Petersen, Silicon as a mechanical material. IEEE Proc, 1982,70(5), 420-457
[32] J.Fujita, S.Mitake, F.Shimizu. Interferometric modulation of an atomic beam by an electric field: a phase hologram for atoms. Physical Review Letters,2000, 84(18): 4027-4030
[33] A. Manz, N. Graber and H.M. Widmer. Miniaturized total chemical analysis systems: A novel concept for chemical sensing.Sensors and Actuators B: Chemical, 1990, 1(6): 244-248
[34]罗怡,娄志峰,褚德南,等.玻璃微流控芯片的制作.纳米技术与精密工程,2004,2 (1) :20-23
[35]罗伟栋. PCR扩增芯片中微加热器结构优化分析.传感技术学报,2005,18 (3) : 627 - 630
[36]孔祥谦.有限单元法在传热学中的应用.北京:科学出版社,1981: 34 - 39
[37]刘海成.MCU-DSP型单片机原理与应用.北京:北就航空航天大学出版社,2006
[38]赵定远,马洪江.16位单片机及语音嵌入式系统.北京:中国水利水电出版本社,2006:1-8
[39]窦日轩.自动控制原理.北京:机械工业出版社,2006
[40]刘丁.自动控制理论.北京:机械工业出版社,2006
[41]陶永华.新型PID控制及其应用.北京:机械工业出版社,2002
[42] Y.Cheng, B.Shew, C.Lin, et al. Ultra-deep LIGA process. Journal of Micromechainics and Microengineering,1999,9(1): 58-63
[43] M.S.Despa , K.W.Kelly , J.R.Collier . Injection molding of polymeric LIGA HARMs. Microsystem Technologies,1999, 6(2): 60-66
[44]程佳,朱纪军,SIMON S.Ang.基于玻璃基底微流控芯片的制备.生物医学工程研究,2005,3(24): 179-182
[45]傅新,刘玲,杨华勇.基于玻璃湿法刻蚀的微流控器件加工工艺研究.机械工程学报,2003 39(11):123-129
[2]马立人,蒋中华.生物芯片.第二版.北京:化学工业出版社,2002
[3]周冬生.生物芯片分类及其技术原理.微生物学免疫学进展,2002 (3):101～107
[4]章春笋,徐进良.连续流动式PCR芯片相关技术研究进展.分析测试学报,2004, 23 (6) :114-118
[5]王廷华,景强.PCR理论与技术.北京:科学出版社,2005
[6] Rabinow, P.Making. PCR: A story of biotechnology. Chicago: University of Chicago Press,1996
[7] Kary B.Mullis .The Unusual Origin of the Polymerase Chain Reaction. Scientific American, 1990,262(4):56-61, 64-5
[8] Mullis,K. , Faloona,F. , Scharf,S. , et al. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symposium in Quantitative Biology ,1986, 51: 263-73
[9] Mullis,K. , Faloona,F. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol ,1987,155: 335-350
[10]周天,邹志清,赵建龙,等. PCR微池芯片的设计、制作及实现样品扩增.功能材料与器件学报,2003, 2 (9):191-194
[11]何文波.基于MEMS技术的PCR生物芯片的研究[硕士学位论文].大连:大连理工大学,2003
[12] A.Rolfs, I.Schuller, U.Finck, I.Weber-Rolfs. PCR: Clinical Diagnostics and Research. New York: Springer-Verlag, 1992
[13] M.J.McPhearson, P.Quirke, and G.R.Taylor. PCR: A Practical Approach. Oxford ,UK: Oxford Univ. Press, 1992
[14] K.B.Mullis, F.Ferte, and R.A.Gibbs.The Polymerase Chain Reaction.Boston: Birkhauser, 1994
[15] E.A.Erlich. PCR Technology: Principles and Applications for DNA Amplification. Oxford,UK: Oxford Univ. Press, 1992
[16] P Wilding, Man A.Shoffner and Larry J.Kricka. PCR in a silicon microstructure.Clin Chem, 1994,40:1815-1818
[17] Zhan Zhao, Zheng Cui, Dafu Cui ,et al. Monolithically integrated PCR biochip for DNA amplification. Sensors and Actuators A, 2003, 108:162-167
[18] Martin U. Kopp, Andrew J.deMello, Andreas Manz ,et al .Chemieal Amplification: Continuous-Flow PCR on a Chip . Science,1998,280(15) : 1046-1048
[19]赵湛,崔大付,于中尧,等.基于MEMS技术的PCR生物芯片的研究.传感器技术,2001,20(6):56-59
[20]崔大付.基于BioMEMS技术的DNA芯片研究.机械强度,2001,23(4):471-475
[21]季旭,刘理大.一种集成PCR反应室与CE的生物芯片的研究.仪器仪表学报,2003,24(4):201-202
[22] Isabel Rodrigue Z, Mariel Lesaieherre. Practical integration of polymerase chain reaction amplification and electrophoretic analysis in microfluidic devices fo genetic analysis. Electrophoresis,2003,24:172-178
[23] Toriello NM, Liu CN, Mathies RA. Mutichannel Reverse Transcription-Polymerase Chain Reaction Microdevice for Rapid Gene Expression and Biomarker Analysis. Analytical Chemistry, 2006, 78 (23):7997-8003
[24] Peng Liu,Tae Seok Seo. Integrated Portable Polymerase Chain Reaction-Capillary Electrophoresis Microsystem for Rapid Forensic Short Tandem Repeat Typing. Analytical Chemistry, 2007, 79 (5):1881-1889
[25] Suzanne Cheng, Russell Higuchi , Mark Stoneking. Complete mitochondrial genome amplication. Nature Genet , 1994 , 7 : 350～351
[26] Nelson W S , Prodohl P A , Avise J C. Development and application of long-PCR for the assay of full-length animal mitochondrial DNA. Mol Ecol , 1996 , 5 : 807～810
[27] Ui Wook Hwang , Chan Jong Park , Tai Soon Yong , et al .One-step PCR amplification of complete arthropod mitochondrial genomes. Molecular Phylogenetics and Evolution , 2001 , 19(3) : 345～352
[28] Iseno K.A novel quantitative PCR with fluorogenic probe.Rinsho Byofi,1997,45(3):218-225
[29] Christin Betz, T.Greg McCollum, Richard T.Mayer.Differential expression of two cinnamate 4-hydroxylase genes in’Valencia’orange(Citrus Sinensis Osbeck).PlantMo1ecular Biology,2001,46(6):741-748
[30] J.Bryzek, K.Petersen, W.McCulley. Micromachines on the March. IEEE Spectrum,1994,31(5),20-31
[31] K.Petersen, Silicon as a mechanical material. IEEE Proc, 1982,70(5), 420-457
[32] J.Fujita, S.Mitake, F.Shimizu. Interferometric modulation of an atomic beam by an electric field: a phase hologram for atoms. Physical Review Letters,2000, 84(18): 4027-4030
[33] A. Manz, N. Graber and H.M. Widmer. Miniaturized total chemical analysis systems: A novel concept for chemical sensing.Sensors and Actuators B: Chemical, 1990, 1(6): 244-248
[34]罗怡,娄志峰,褚德南,等.玻璃微流控芯片的制作.纳米技术与精密工程,2004,2 (1) :20-23
[35]罗伟栋. PCR扩增芯片中微加热器结构优化分析.传感技术学报,2005,18 (3) : 627 - 630
[36]孔祥谦.有限单元法在传热学中的应用.北京:科学出版社,1981: 34 - 39
[37]刘海成.MCU-DSP型单片机原理与应用.北京:北就航空航天大学出版社,2006
[38]赵定远,马洪江.16位单片机及语音嵌入式系统.北京:中国水利水电出版本社,2006:1-8
[39]窦日轩.自动控制原理.北京:机械工业出版社,2006
[40]刘丁.自动控制理论.北京:机械工业出版社,2006
[41]陶永华.新型PID控制及其应用.北京:机械工业出版社,2002
[42] Y.Cheng, B.Shew, C.Lin, et al. Ultra-deep LIGA process. Journal of Micromechainics and Microengineering,1999,9(1): 58-63
[43] M.S.Despa , K.W.Kelly , J.R.Collier . Injection molding of polymeric LIGA HARMs. Microsystem Technologies,1999, 6(2): 60-66
[44]程佳,朱纪军,SIMON S.Ang.基于玻璃基底微流控芯片的制备.生物医学工程研究,2005,3(24): 179-182
[45]傅新,刘玲,杨华勇.基于玻璃湿法刻蚀的微流控器件加工工艺研究.机械工程学报,2003 39(11):123-129