基于MEMS技术的PCR芯片的研究
|
摘要
PCR芯片是对微量DNA进行扩增放大的一种生化反应芯片，在整个生物芯片的研究和发展中起着重要的作用。综合国内外在PCR芯片研究方面取得的成果，采用基于MEMS技术的研究方法，在硅基片上实现了集成的PCR芯片。芯片上集成了DNA反应室，微通道、温度传感器以及加热体等功能和部件，大小为8mm×4mm×0.3mm。利用深刻蚀技术制作出一个4mm×2mm×0.3mm容积约为2微升的反应室，反应室的底为约2微米厚的氮化硅(Si_3N_4)膜。铂加热子和温度传感器是在Si_3N_4膜上沉积和制作出图形形成的。这样的PCR芯片可以使系统微型化，可以很方便地应用在医疗诊断、野外生化分析等领域。配合PCR芯片研制了微型热循环控制系统，包括硬件和软件两大部分。硬件的设计主要从减少尺度，降低功耗等方面考虑，以实现和PCR芯片相适应的仪器微型化，从而达到便携、快捷地实现DNA—PCR的反应。软件部分从温度控制精度、以硬件简捷、操作以人为本的原则进行编制。研究设计了不同PCR芯片的结构和加热子等的热传导、温度场分布等物理特性。从理论上证明设计的可行性。进行了PCR芯片的实验前的表面处理方法的实验，选用乙肝病毒的试剂盒，进行了芯片上的DNA扩增实验。DNA利用SYBR@GreenⅠ荧光染料进行标记，扩增的产物在荧光显微镜下用Cool CCD记录，取得了满意的结果。PCR芯片的物理特性如下：1．反应池容积：＜2μl；2．升温速率：可达15℃／s；3．降温速率：可达10℃／s；4．控温精度：≤0.4℃；5．功耗：＜800mW。
DNA PCR amplification is bioreaction for DNA analysis. It expands a little to a large amount of DNA molecule. Reviewing the research on the PCR biochip, one monolithically integrated was designed and fabricated in a substrate based on MEMS technology. DNA reaction chamber, microchannel, temperature sensor and heater are integrated on the PCR biochip. The chip PCR is realized with the material of silicon with the size of 8mm ×4mm ×0.3mm. The volume of chamber is roughly 2 micro liters with the size of 4mm×2mm×0.3mm.The bottom of chamber is constructed by the film of Si3N4 with the thickness of 2 micrometers. The platinum heater and temperature sensor are deposited on the Si3N4 film. With the PCR biochip, a miniaturized analyzing system can be realized for PCR, and it can be very expediently applied to the medical, environment analysis in outdoor. Matching with the PCR biochip, a miniaturized heat
cycler system is designed and fabricated with proper hardware and software.The design of the hardware is based on the principle of reducing the dimensions, lowering the power consumption, fabricating a pocketable instrument. The software is a key part for accurately control the temperature and easy operation etc. In the paper, PCR biochips with different structrues were designed and researched in the respects of its physical characteristics including thermal transfer of the heater, the temperature field distribution etc. A standard Hepatitis B (HB) testing reagent was used as thermal cycling materials for DNA amplification with the PCR biochip. The experiment employed the fluorescent dye S YBR Green I assay technique so that a fluorescent can be detected. The test has been completed with enhanced fluorescence result when 30 cycles finished on the PCR biochip. The PCR biochip's characteristic is as follows: 1.chamber capacity
:<2 μl. 2. heating rate 15℃/s. 3.cooling rate 10 ℃/s. 4. temperature accuracy ≤0.4 ℃. 5. power consumption <800 mW.
DNA PCR amplification is bioreaction for DNA analysis. It expands a little to a large amount of DNA molecule. Reviewing the research on the PCR biochip, one monolithically integrated was designed and fabricated in a substrate based on MEMS technology. DNA reaction chamber, microchannel, temperature sensor and heater are integrated on the PCR biochip. The chip PCR is realized with the material of silicon with the size of 8mm ×4mm ×0.3mm. The volume of chamber is roughly 2 micro liters with the size of 4mm×2mm×0.3mm.The bottom of chamber is constructed by the film of Si3N4 with the thickness of 2 micrometers. The platinum heater and temperature sensor are deposited on the Si3N4 film. With the PCR biochip, a miniaturized analyzing system can be realized for PCR, and it can be very expediently applied to the medical, environment analysis in outdoor. Matching with the PCR biochip, a miniaturized heat
cycler system is designed and fabricated with proper hardware and software.The design of the hardware is based on the principle of reducing the dimensions, lowering the power consumption, fabricating a pocketable instrument. The software is a key part for accurately control the temperature and easy operation etc. In the paper, PCR biochips with different structrues were designed and researched in the respects of its physical characteristics including thermal transfer of the heater, the temperature field distribution etc. A standard Hepatitis B (HB) testing reagent was used as thermal cycling materials for DNA amplification with the PCR biochip. The experiment employed the fluorescent dye S YBR Green I assay technique so that a fluorescent can be detected. The test has been completed with enhanced fluorescence result when 30 cycles finished on the PCR biochip. The PCR biochip's characteristic is as follows: 1.chamber capacity
:<2 μl. 2. heating rate 15℃/s. 3.cooling rate 10 ℃/s. 4. temperature accuracy ≤0.4 ℃. 5. power consumption <800 mW.
引文
1. http://www.colband.com.br/ativ/nete/biot/textos/biocomputadores/002.htm.
2. Robert F. Service, Microchip Arrays Put DNA on the Spot Science 1998 October 16; 282: 396-399.
3. Robert F. Service, Coming Soon: The Pocket DNA Sequencer Science 1998 October 16; 282: 399-401.
4. Schena M, Heller RA, Theriault TP, Konrad K, Lachenmeier E, Davis RW. 1998. Microarrays: biotechnology's discovery platform for functional genomics. Trends Biotechnol 1998 Jul; 16(7):301-306
5. Robert J. Lipshutz, Stephen P.A. Fodor, Thomas R. Gingeras & David J. Lockhart, High density synthetic oligonucleotide arrays,Nature genetics supplement volume 21·january 1999,20-24
6. http://www.lmbe. seu.edu.cn/neirong/neirongl/jiyin1.htm
7. Gregory T. A. Kovacs, Kurt Petersen and Michael Albin. Silicon Micromachining, Anal. Chem. 1996, 68, 407A-412A
8. N. T. Nguyen, X. Y. Huang: Miniature Valveless Pumps Based on Printed Circuit Board Technique, Sensors and Actuators A , Volume 88/2, Elsevier, 2001, pp. 104-111.
9. Bruno Michel, Printing Meets Lithography, The Industrial Physicist, AUGUST/SEPTEMBER 2002,16-19
10. Marshall, A.; Hodgson, J. DNA chips - an array of possibilities. Nature Biotechnology 1998, 16(1), 27-31.
11. Gwynne P. and Page G. Microarray analysis: the next revolution in molecular biology. Science, 1999 August 6.
12. Walt DR. Bead-based Fiber-Optic Arrays. Science, 2000 January 21; 287: 451-452.
13. Khan J, Saal LH, Bittner ML, Chen Y, Trent JM, Meltzer PS. Expression profiling in cancer using cDNA microarrays. Electrophoresis 1999 Feb;20(2):223-9
14. Baldwin D, Crane V, Rice D. A comparison of gel-based, nylon filter and microarray techniques to detect differential RNA expression in plants. Curr Opin Plant Biol1999 Apr;2(2):96-103
15. Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, Torhorst J, Mihatsch MJ, Sauter G, Kallioniemi OP. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med 1998 Jul;4(7):844-847
16. G. MacBeath and S.L. Schreiber, Printing Proteins as Microarrays for High-Throughput Function Determination, Science 2000 September 8; 289(5485):
p. 1760-1763.
17. LJ Kricka, "Miniaturization of Analytical Systems," Clinical Chemistry 44 (1998): 2008-2014.
18. Sanders GHW, Manz A, Chip-based microsystems for gernomic and proteomic analysis. Tredds Anal Chem 2000,19, 364-378.
19. Anderson RC, Su X, Bogdan GJ, Fenton J, A miniature integrated device for automated multistep genetic assays, Nucleric Acids Res 2000, 28, e60.
20. Chou H,Spence C, Scherer A, Quake S, A microfabricated device for sizing and sorting DNA molecules, Proc Natl Acad Sci USA 1999,96, 11-13.
21. Jakeway SC, Mello AJ,Russell EL, Miniaturized total analysis systems for biological analysis, Fresenius Janal Chem 2000, 366, 525-539.
22. Rolfe C. Anderson, Xing Su, Gregory J. Bogdan and Jeffery Fenton A miniature integrated device for automated multistep genetic assays Nucleic Acids Research, 2000, Vol. 28, No. 12 E60-e60
23. Mark A. Burns, Brian N. Johnson, Sundaresh N. Brahraasandra, Kalyan Handique, James R. Webster, Madhavi Krishnan, Timothy S. Sammarco, Piu M. Man, Darren Jones, Dylan Heldsinger, Carlos H. Mastrangelo, David T. Burke An Integrated Nanoliter DNA Analysis Device Science, Vol. 282, 16 Oct 1998, pp. 484 - 487
24. K. B. Mullis, "The unusual origin of the polymerase chain reaction," Scientific American, vol. 270, pp. 56-65, 1990.
25. A. Rolfs, I. Schuller, U. Finck, and I. Weber-Rolls, PCR: Clinical Diagnostics and Research. New York: Springer-Verlag, 1992.
26. M. J. McPhearson, P. Quirke, and G. R. Taylor, PCR: A Practical Approach, Vol. 1. Oxford, UK: Oxford Univ. Press, 1992.
27. K. B. Mullis, F. Ferre, and R. A. Gibbs, the Polymerase Chain Reaction. Boston: Birkhauser, 1994.
28. E. A. Erliech, PCR Technology: Principles and Applications for DNA Amplification. Oxford, UK: Oxford Univ. Press, 1992.
29. P. L. Crotty, R. A. Staggs, P. T Porter, A. A. Killen, and R. C. McGleenen, "Quantitative analysis in molecular diagnostics," Human Pathology, vol. 25, pp. 572-579, 1994.
30. F. Ferre, "Quantitative or semi-quantitative PCR: reality vs. myth," PCR Meth. Appl., vol. 2, pp. 1-9, 1992.
31．保罗·拉比诺 著，朱玉贤译，PCR传奇—一个生物技术的故事，上海科技教育出版社，1998年12月。
32. A. T. Andrews, Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications. New York: Oxford, 1986.
33. P. D. Grossman and J. C. Colburn, Capillary Electrophoresis: Theory and Practice. New York: Academic Press, 1992.
34. A. E. Karger, J. T. Ives, R. B. Weiss, J. M. Harris, and R. F. Gesteland, "Imaging of fluorescent and chemiluminescent DNA hybrids using a 2-D CCD camera," in Proc. SPIE Conf. New Technologies in Cytometry and Molecular Biology, vol.
1206, pp. 78-89, 1991.
35. A. E. Karger, R.Weiss, and R. F. Gesteland, "Digital chemiluminescence imaging of DNA sequencing blots using a charge-coupled device camera," Nucleic Acids Res., vol. 20, pp. 6657-6665, 1992.
36. J. H. Kenten, J. Casadei, J. Link, S. Lupold, J. Willey, M. Powell, A. Rees, and R. Massey, "Rapid electrochemiluminescence assays of polymerase chain reaction products," Clin. Chem., vol. 37, pp. 1626-1632, 1991.
37. H. Yu, J. G. Bruno, C. T. Chang, J. J. Calomiris, M. T. Goode, and D. L. Gatto-Menking, "A compartive study of PCR product detection and quantitation by electro-chemiluminescence and fluorescence," J. Biolumin. Chemilum., vol. 10, pp. 239-245, 1995.
38. G. T. Walker, C. M. Little, J. G. Nadeau, and D. D. Shank, "Isothermal in vitro amplification of DNA by arestriction enzyme/DNA polymerase system," Proc. Natl. Acad. Sci. USA, vol. 89, pp. 392-396, 1992.
39. J.Bryzek, K.Petersen, W. McCulley, Micromachines on the March, IEEE Spect. 31 (5),20, 1994.
40. K. Petersen, Silicon as a mechanical material, IEEE Proc. 70, 420, 1982.
41. M. L. Roukes, NANOELECTROMECHANICAL SYSTEMS, Solid-State Sensor and Actuator Workshop Hi/ton Head Island, South Carolina, June 4-8, 2000 367-376
42. Soon-Soo Park, Dong-Il Park, Sung-Ho Hahm, Jong-Hyun Lee, Hyun-Chul Choi, and Jung-Hee Lee, Fabrication of a Lateral Field Emission Triode witha High Current Density and High Transconductance Using the Local Oxidation of the Polysilicon Layer, IEEE TRANSACTIONS ON ELECTRON DEVICES, VOL. 46, NO. 6, JUNE 1999 1283-1289
43. 刘静，《微米／纳米尺度传热学》 科学出版社，2001
44. J. J. Carrino and H. H. Lee, "Nucleic acid amplification methods," J. Microb. Meth., vol. 23, pp. 3-20, 1995.
45. M. A. Northrup, M. T. Ching, R. M. White, and R. T. Watson, "DNA amplification with a microfabricated reaction chamber," in Proc. 1993 IEEE Int. Conf. Solid-State Sens. Actuat., pp. 924-926, 1993.
46. M. A. Northrup, C. Gonzalez, D. Hadley, R. E Hills, P. Lander, S. Lehew, R. Saiki, J. J. Sninsky, and R. Watson, "A MEMS-based miniature DNA analysis system," in Proc. 1995 IEEE Int. Conf. Solid-State Sens. Actuat., pp. 764-767, 1995.
47. Martin U. Kopp, Andrew J. de Mello, Andreas Manz Chemical Amplification: Continuous-Flow PCR on a Chip Science Volume 280, Number 5366, Issue of 15 May 1998, pp. 1046-1048.
48. Jian Liu, Markus Enzelberger, Stephen Quake, A nanpliter rotary device for polymerase chain reaction, Electrophoresis 2002, 23, 1531-1536.
49. Hidenori Nagai, Yuji Murakami, Kenji Yokoyama and Eiichi Tamiya, High-throughput PCR in silicon based microchamber array, Biosensors and Bioelectronics, Volume 16, Issues 9-12, December 2001, Pages 1015-1019.
50. Julia Khandurina, Timothy E. McKnight, Stephen C. Jacobson, Larry C. Waters,
Robert S. Foote, and J. Michael Ramsey, Integrated system for rapid PCR-based DNA analysis in Microfluidic Devices. Anal. Chem. 2000, 72, 2995-3000
51. Jong Week Hong, Teruo Fujii, Minoru Seki, Takatoki Yamamoto, Isao Endo, Integration of gene amplification and capillary gel electrophoresis on a polydimethylsiloxane-glass hybrid microchip. Electrophoresis 2001, 22,328-333
52. http://faculty. virginia.edu/landers/IRframe.htm
53. http://www.beckman.com/resourcecenter/labresources/ce/cedefinitionmodes.asp
54. M.Frenea;H.Lhermite;B.L.Pioufle;H.Fujita, Design of biochip microelectrode arrays for cell arrangement, annual international IEEE-EMBS Special topic conference on microtechnologies in medecine & biology,May,2OO2,page 140～143
55. Fernandez-Morales F.;J. Samitier;O.Ruiz;J.Bausells;E.Abdelhamid, Design and simulation of a dielectrophoretic-based microsystem for bioparticle handling, Annual international IEEE-EMBS special topic conference on microtechnologies in medicine & biology, Oct,2OOO,page 429～433
56. H.Sano;H.Kabata;O.Kurosawa;M.Washizu, Dielectrophoretic chromatography with cross-flow injection, Micro Electro Mechanical Systems,2OO2,page 11～14
57. A. T. Woolley and R. A. Mathies, Ultra-high-speed DNA fragment separations using microfabricated capillary array electrophoresis chips, Anal. Chem., vol. 67, pp. 3676-3680, 1995.
58. http://www.lightcycler-online.com/lc_principles/lc_prin_dna_det01.htm
59. C G J Schabmueller, J R Pollard, A G R Evans, J S Wilkinson, G Ensell and A Brunnschweiler. Integrated diode detector and optieal fibres for in situ detection within micromachined polymerase chain reaction chips J. Micromech. Microeng. 11 (2001) 329-333
60. Jing Gaoshan, Zhang Jian, Zhu Xiaoshan, Feng Jihong, Tan Zhimin, Liu Litian, Cheng Jing, Temperature Control System for Biochemical Reactions in Microchip-Based Devices, TSINGHUA SCIENCE and TECHNOLOGY, Vol 6, No 3, August 2001, pp269-272
61. http://www. ime.org.sg/mems/mems_biochip.htm
62. Eric T. Lagally, Peter C. Simpson, Richard A. Mathies, Monolithic integrated microfluidic DNA amplification and capillary electrophoresis analysis system . Sensors and Actuators, B 63 2000, pp138-146
63. Larry C. Waters, Stephen C. Jacobsen, Natalia Kroutchinina, Julia Khandurina, Robert S. Foote, and J. Michael Ramsey, Multiple Sample PCR Amplification and Electrophoretic Analysis on a Microchip, Anal. Chem. 1998,70,5172-5176.
64. Adam T. Woolley, Dean Hadley, Phoeb E Landre, Andrew J. DeMello, Richard A. Mathies, and M. Allen Northrup, Functional Integration of PCR Amplification and Caplliary Electrophoresis in a Microfabricated DNA Analysis Device. Anal. Chem. 1996, 68, 4081-4086.
65. Ajit M. Chaudhari, Timothy M. Woudenberg, Michael Albin, and Kenneth E. Goodson, Transient Liquid Crystal Thermometry of Microfabricated PCR vessel Arrays, J. Microelectromechanical systems, Vol 7, No. 4 Dec. 1998, 345-355
66. Huinan Yu , Palaniappan Sethu , Tony Chan , Natalia Kroutchinina, Jocquese
Blackwell, Carlos H. Mastrangelo, and Piotr Grodzinski, a miniaturized and integrated plastic thermal chemical reactor for genetic analysis, Micro Total Analysis Systems 2000 Conference, Enschede, Netherlands, May 14-18, 2000 pp. 545-548.
67. Stephen A. Campbell, The Science and Engineering of Microelectronic Fabrication, Oxford Unicersity Press. Inc. 2001
68. http://roboflag.carleton.ca/resources/technical/pic_introduction, shtml
69．王之芳，《传感器应用技术》，西北工业大学出版社，1991年12月第1版，第195页
70．张琳娜 刘武发，《传感检测技术及应用》，中国计量出版社，1999年12月第1版，第142页
71．杨世铭，陶文铨，《传热学》，高等教育出版社，1998
72. T. B. Taylor, E. S.Winn-Deen, E. Picozza, T. M.Woudenberg, and M. Albin, "Optimization of the performance of the polymerase chain reaction in silicon-based microstructures," Nucleic Acids Res., vol. 25, pp. 3164-3168, 1997.
73. Mann A. Shoffner, Jing Cheng, Georgi E. Hvichia, Larry J. Krieka and Peter Wilding, Chip PCR. I. Surface passivation of microfabricated silicon-glass chips for PCR , Nucleic Acids Research, 1996 Vol. 24, 375-379.
74. Joel S. Rossier, Rosaria Ferrigno, Hubert H. Girault, Electrophoresis with electrochemical detection in a polymer microdevice, J. electroanalytical chemistry, 492 (2000) 15-22
75. Yong Zeng, Hong Chen, Dai-wen Pang, Zong-li Wang, and Jie-ke Cheng, Microchip capillary electrophoresis with electrochemical detection, Anal Chem. 2002, 74, 2441-2445
76. R. Scott Martin, Andrew J. Gawron, and Susan M. Lunte, Dual-electrode electrochemical detection for poly(dimethylsiloxane) fabricated capillary electrophoresis microchip. Anal. Chem. 2000, 72, 3196-3202
77. Benedikt Fraβ, Andreas Neyer, Matthias Johnck, Dirk Siepe, Friedhelm Eisenbeiβ, Gunther Weber, Roland Hergenroder, A new PMMA microchip device for isotachophoresis with integrated conductivity detector, Sensors and Actuators B 72 (2001) 249-258
78. Woolley AT, Lao KQ, Glazer AN, Mathies RA, Capillary electrophoresis chips with integrated electrochemical detection, Anal Chem. 1998 70, 684-688
2. Robert F. Service, Microchip Arrays Put DNA on the Spot Science 1998 October 16; 282: 396-399.
3. Robert F. Service, Coming Soon: The Pocket DNA Sequencer Science 1998 October 16; 282: 399-401.
4. Schena M, Heller RA, Theriault TP, Konrad K, Lachenmeier E, Davis RW. 1998. Microarrays: biotechnology's discovery platform for functional genomics. Trends Biotechnol 1998 Jul; 16(7):301-306
5. Robert J. Lipshutz, Stephen P.A. Fodor, Thomas R. Gingeras & David J. Lockhart, High density synthetic oligonucleotide arrays,Nature genetics supplement volume 21·january 1999,20-24
6. http://www.lmbe. seu.edu.cn/neirong/neirongl/jiyin1.htm
7. Gregory T. A. Kovacs, Kurt Petersen and Michael Albin. Silicon Micromachining, Anal. Chem. 1996, 68, 407A-412A
8. N. T. Nguyen, X. Y. Huang: Miniature Valveless Pumps Based on Printed Circuit Board Technique, Sensors and Actuators A , Volume 88/2, Elsevier, 2001, pp. 104-111.
9. Bruno Michel, Printing Meets Lithography, The Industrial Physicist, AUGUST/SEPTEMBER 2002,16-19
10. Marshall, A.; Hodgson, J. DNA chips - an array of possibilities. Nature Biotechnology 1998, 16(1), 27-31.
11. Gwynne P. and Page G. Microarray analysis: the next revolution in molecular biology. Science, 1999 August 6.
12. Walt DR. Bead-based Fiber-Optic Arrays. Science, 2000 January 21; 287: 451-452.
13. Khan J, Saal LH, Bittner ML, Chen Y, Trent JM, Meltzer PS. Expression profiling in cancer using cDNA microarrays. Electrophoresis 1999 Feb;20(2):223-9
14. Baldwin D, Crane V, Rice D. A comparison of gel-based, nylon filter and microarray techniques to detect differential RNA expression in plants. Curr Opin Plant Biol1999 Apr;2(2):96-103
15. Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, Torhorst J, Mihatsch MJ, Sauter G, Kallioniemi OP. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med 1998 Jul;4(7):844-847
16. G. MacBeath and S.L. Schreiber, Printing Proteins as Microarrays for High-Throughput Function Determination, Science 2000 September 8; 289(5485):
p. 1760-1763.
17. LJ Kricka, "Miniaturization of Analytical Systems," Clinical Chemistry 44 (1998): 2008-2014.
18. Sanders GHW, Manz A, Chip-based microsystems for gernomic and proteomic analysis. Tredds Anal Chem 2000,19, 364-378.
19. Anderson RC, Su X, Bogdan GJ, Fenton J, A miniature integrated device for automated multistep genetic assays, Nucleric Acids Res 2000, 28, e60.
20. Chou H,Spence C, Scherer A, Quake S, A microfabricated device for sizing and sorting DNA molecules, Proc Natl Acad Sci USA 1999,96, 11-13.
21. Jakeway SC, Mello AJ,Russell EL, Miniaturized total analysis systems for biological analysis, Fresenius Janal Chem 2000, 366, 525-539.
22. Rolfe C. Anderson, Xing Su, Gregory J. Bogdan and Jeffery Fenton A miniature integrated device for automated multistep genetic assays Nucleic Acids Research, 2000, Vol. 28, No. 12 E60-e60
23. Mark A. Burns, Brian N. Johnson, Sundaresh N. Brahraasandra, Kalyan Handique, James R. Webster, Madhavi Krishnan, Timothy S. Sammarco, Piu M. Man, Darren Jones, Dylan Heldsinger, Carlos H. Mastrangelo, David T. Burke An Integrated Nanoliter DNA Analysis Device Science, Vol. 282, 16 Oct 1998, pp. 484 - 487
24. K. B. Mullis, "The unusual origin of the polymerase chain reaction," Scientific American, vol. 270, pp. 56-65, 1990.
25. A. Rolfs, I. Schuller, U. Finck, and I. Weber-Rolls, PCR: Clinical Diagnostics and Research. New York: Springer-Verlag, 1992.
26. M. J. McPhearson, P. Quirke, and G. R. Taylor, PCR: A Practical Approach, Vol. 1. Oxford, UK: Oxford Univ. Press, 1992.
27. K. B. Mullis, F. Ferre, and R. A. Gibbs, the Polymerase Chain Reaction. Boston: Birkhauser, 1994.
28. E. A. Erliech, PCR Technology: Principles and Applications for DNA Amplification. Oxford, UK: Oxford Univ. Press, 1992.
29. P. L. Crotty, R. A. Staggs, P. T Porter, A. A. Killen, and R. C. McGleenen, "Quantitative analysis in molecular diagnostics," Human Pathology, vol. 25, pp. 572-579, 1994.
30. F. Ferre, "Quantitative or semi-quantitative PCR: reality vs. myth," PCR Meth. Appl., vol. 2, pp. 1-9, 1992.
31．保罗·拉比诺 著，朱玉贤译，PCR传奇—一个生物技术的故事，上海科技教育出版社，1998年12月。
32. A. T. Andrews, Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications. New York: Oxford, 1986.
33. P. D. Grossman and J. C. Colburn, Capillary Electrophoresis: Theory and Practice. New York: Academic Press, 1992.
34. A. E. Karger, J. T. Ives, R. B. Weiss, J. M. Harris, and R. F. Gesteland, "Imaging of fluorescent and chemiluminescent DNA hybrids using a 2-D CCD camera," in Proc. SPIE Conf. New Technologies in Cytometry and Molecular Biology, vol.
1206, pp. 78-89, 1991.
35. A. E. Karger, R.Weiss, and R. F. Gesteland, "Digital chemiluminescence imaging of DNA sequencing blots using a charge-coupled device camera," Nucleic Acids Res., vol. 20, pp. 6657-6665, 1992.
36. J. H. Kenten, J. Casadei, J. Link, S. Lupold, J. Willey, M. Powell, A. Rees, and R. Massey, "Rapid electrochemiluminescence assays of polymerase chain reaction products," Clin. Chem., vol. 37, pp. 1626-1632, 1991.
37. H. Yu, J. G. Bruno, C. T. Chang, J. J. Calomiris, M. T. Goode, and D. L. Gatto-Menking, "A compartive study of PCR product detection and quantitation by electro-chemiluminescence and fluorescence," J. Biolumin. Chemilum., vol. 10, pp. 239-245, 1995.
38. G. T. Walker, C. M. Little, J. G. Nadeau, and D. D. Shank, "Isothermal in vitro amplification of DNA by arestriction enzyme/DNA polymerase system," Proc. Natl. Acad. Sci. USA, vol. 89, pp. 392-396, 1992.
39. J.Bryzek, K.Petersen, W. McCulley, Micromachines on the March, IEEE Spect. 31 (5),20, 1994.
40. K. Petersen, Silicon as a mechanical material, IEEE Proc. 70, 420, 1982.
41. M. L. Roukes, NANOELECTROMECHANICAL SYSTEMS, Solid-State Sensor and Actuator Workshop Hi/ton Head Island, South Carolina, June 4-8, 2000 367-376
42. Soon-Soo Park, Dong-Il Park, Sung-Ho Hahm, Jong-Hyun Lee, Hyun-Chul Choi, and Jung-Hee Lee, Fabrication of a Lateral Field Emission Triode witha High Current Density and High Transconductance Using the Local Oxidation of the Polysilicon Layer, IEEE TRANSACTIONS ON ELECTRON DEVICES, VOL. 46, NO. 6, JUNE 1999 1283-1289
43. 刘静，《微米／纳米尺度传热学》 科学出版社，2001
44. J. J. Carrino and H. H. Lee, "Nucleic acid amplification methods," J. Microb. Meth., vol. 23, pp. 3-20, 1995.
45. M. A. Northrup, M. T. Ching, R. M. White, and R. T. Watson, "DNA amplification with a microfabricated reaction chamber," in Proc. 1993 IEEE Int. Conf. Solid-State Sens. Actuat., pp. 924-926, 1993.
46. M. A. Northrup, C. Gonzalez, D. Hadley, R. E Hills, P. Lander, S. Lehew, R. Saiki, J. J. Sninsky, and R. Watson, "A MEMS-based miniature DNA analysis system," in Proc. 1995 IEEE Int. Conf. Solid-State Sens. Actuat., pp. 764-767, 1995.
47. Martin U. Kopp, Andrew J. de Mello, Andreas Manz Chemical Amplification: Continuous-Flow PCR on a Chip Science Volume 280, Number 5366, Issue of 15 May 1998, pp. 1046-1048.
48. Jian Liu, Markus Enzelberger, Stephen Quake, A nanpliter rotary device for polymerase chain reaction, Electrophoresis 2002, 23, 1531-1536.
49. Hidenori Nagai, Yuji Murakami, Kenji Yokoyama and Eiichi Tamiya, High-throughput PCR in silicon based microchamber array, Biosensors and Bioelectronics, Volume 16, Issues 9-12, December 2001, Pages 1015-1019.
50. Julia Khandurina, Timothy E. McKnight, Stephen C. Jacobson, Larry C. Waters,
Robert S. Foote, and J. Michael Ramsey, Integrated system for rapid PCR-based DNA analysis in Microfluidic Devices. Anal. Chem. 2000, 72, 2995-3000
51. Jong Week Hong, Teruo Fujii, Minoru Seki, Takatoki Yamamoto, Isao Endo, Integration of gene amplification and capillary gel electrophoresis on a polydimethylsiloxane-glass hybrid microchip. Electrophoresis 2001, 22,328-333
52. http://faculty. virginia.edu/landers/IRframe.htm
53. http://www.beckman.com/resourcecenter/labresources/ce/cedefinitionmodes.asp
54. M.Frenea;H.Lhermite;B.L.Pioufle;H.Fujita, Design of biochip microelectrode arrays for cell arrangement, annual international IEEE-EMBS Special topic conference on microtechnologies in medecine & biology,May,2OO2,page 140～143
55. Fernandez-Morales F.;J. Samitier;O.Ruiz;J.Bausells;E.Abdelhamid, Design and simulation of a dielectrophoretic-based microsystem for bioparticle handling, Annual international IEEE-EMBS special topic conference on microtechnologies in medicine & biology, Oct,2OOO,page 429～433
56. H.Sano;H.Kabata;O.Kurosawa;M.Washizu, Dielectrophoretic chromatography with cross-flow injection, Micro Electro Mechanical Systems,2OO2,page 11～14
57. A. T. Woolley and R. A. Mathies, Ultra-high-speed DNA fragment separations using microfabricated capillary array electrophoresis chips, Anal. Chem., vol. 67, pp. 3676-3680, 1995.
58. http://www.lightcycler-online.com/lc_principles/lc_prin_dna_det01.htm
59. C G J Schabmueller, J R Pollard, A G R Evans, J S Wilkinson, G Ensell and A Brunnschweiler. Integrated diode detector and optieal fibres for in situ detection within micromachined polymerase chain reaction chips J. Micromech. Microeng. 11 (2001) 329-333
60. Jing Gaoshan, Zhang Jian, Zhu Xiaoshan, Feng Jihong, Tan Zhimin, Liu Litian, Cheng Jing, Temperature Control System for Biochemical Reactions in Microchip-Based Devices, TSINGHUA SCIENCE and TECHNOLOGY, Vol 6, No 3, August 2001, pp269-272
61. http://www. ime.org.sg/mems/mems_biochip.htm
62. Eric T. Lagally, Peter C. Simpson, Richard A. Mathies, Monolithic integrated microfluidic DNA amplification and capillary electrophoresis analysis system . Sensors and Actuators, B 63 2000, pp138-146
63. Larry C. Waters, Stephen C. Jacobsen, Natalia Kroutchinina, Julia Khandurina, Robert S. Foote, and J. Michael Ramsey, Multiple Sample PCR Amplification and Electrophoretic Analysis on a Microchip, Anal. Chem. 1998,70,5172-5176.
64. Adam T. Woolley, Dean Hadley, Phoeb E Landre, Andrew J. DeMello, Richard A. Mathies, and M. Allen Northrup, Functional Integration of PCR Amplification and Caplliary Electrophoresis in a Microfabricated DNA Analysis Device. Anal. Chem. 1996, 68, 4081-4086.
65. Ajit M. Chaudhari, Timothy M. Woudenberg, Michael Albin, and Kenneth E. Goodson, Transient Liquid Crystal Thermometry of Microfabricated PCR vessel Arrays, J. Microelectromechanical systems, Vol 7, No. 4 Dec. 1998, 345-355
66. Huinan Yu , Palaniappan Sethu , Tony Chan , Natalia Kroutchinina, Jocquese
Blackwell, Carlos H. Mastrangelo, and Piotr Grodzinski, a miniaturized and integrated plastic thermal chemical reactor for genetic analysis, Micro Total Analysis Systems 2000 Conference, Enschede, Netherlands, May 14-18, 2000 pp. 545-548.
67. Stephen A. Campbell, The Science and Engineering of Microelectronic Fabrication, Oxford Unicersity Press. Inc. 2001
68. http://roboflag.carleton.ca/resources/technical/pic_introduction, shtml
69．王之芳，《传感器应用技术》，西北工业大学出版社，1991年12月第1版，第195页
70．张琳娜 刘武发，《传感检测技术及应用》，中国计量出版社，1999年12月第1版，第142页
71．杨世铭，陶文铨，《传热学》，高等教育出版社，1998
72. T. B. Taylor, E. S.Winn-Deen, E. Picozza, T. M.Woudenberg, and M. Albin, "Optimization of the performance of the polymerase chain reaction in silicon-based microstructures," Nucleic Acids Res., vol. 25, pp. 3164-3168, 1997.
73. Mann A. Shoffner, Jing Cheng, Georgi E. Hvichia, Larry J. Krieka and Peter Wilding, Chip PCR. I. Surface passivation of microfabricated silicon-glass chips for PCR , Nucleic Acids Research, 1996 Vol. 24, 375-379.
74. Joel S. Rossier, Rosaria Ferrigno, Hubert H. Girault, Electrophoresis with electrochemical detection in a polymer microdevice, J. electroanalytical chemistry, 492 (2000) 15-22
75. Yong Zeng, Hong Chen, Dai-wen Pang, Zong-li Wang, and Jie-ke Cheng, Microchip capillary electrophoresis with electrochemical detection, Anal Chem. 2002, 74, 2441-2445
76. R. Scott Martin, Andrew J. Gawron, and Susan M. Lunte, Dual-electrode electrochemical detection for poly(dimethylsiloxane) fabricated capillary electrophoresis microchip. Anal. Chem. 2000, 72, 3196-3202
77. Benedikt Fraβ, Andreas Neyer, Matthias Johnck, Dirk Siepe, Friedhelm Eisenbeiβ, Gunther Weber, Roland Hergenroder, A new PMMA microchip device for isotachophoresis with integrated conductivity detector, Sensors and Actuators B 72 (2001) 249-258
78. Woolley AT, Lao KQ, Glazer AN, Mathies RA, Capillary electrophoresis chips with integrated electrochemical detection, Anal Chem. 1998 70, 684-688