臺灣博碩士論文加值系統

近十幾年來，由於微機電技術愈來愈成熟，應用面也隨之愈來愈廣，如光學、能源、感測元件、生物等各方面領域皆有出色的研究發展。然後隨著生活品質的提升，人類在醫療方面的要求提高，因此微機電應用於生物技術方面潛力無窮，如複製晶片、檢測晶片、分離晶片等相當多的文章發表。
然而本論文針對生物技術中DNA複製與檢測進行研究，利用微機電加工技術進行設計與製作具有快速聚合酶連鎖反應(PCR)元件，以及無需螢光標地的微懸臂樑陣列DNA檢測晶片。
在DNA複製研究方面，提出一種旋轉機構方式，讓DNA複製檢體可快速進行不同溫度間轉換，有效減少聚合酶連鎖反應熱循環時間。過程中利用CFD ACE+軟體進行DNA腔體溫度及溫度轉換時間的模擬。利用金屬沈積技術製作薄膜加熱器，使用KOH蝕刻製作DNA腔體，整體平台完成後，以大腸桿菌DNA為例完成100bp複製動作。
在微懸臂樑陣列DNA檢測晶片方面，進行設計與製作出微懸臂樑陣列結構。研究中提出以SU-8光阻進行鑄模製造方式，有別於傳統的表面加工或體型加工。並且將所製作出微懸臂樑陣列整合於PDMS流道內完成檢測晶片。檢測過程方面分別利用螢光檢測方式和自行架設之光學位移量測平台進行大腸桿菌DNA探針鍵結以及互補雜交於懸臂樑表面之檢測。

In recent years, MEMS technology have been developed and applied in various kinds of fields, such as, optics, power, sensor, bioengineer and etc. However, with the improvement of quality of the life, people want to have better medical treatment. MEMS technology is applied by biotechnology such as PCR chip, detection chip, separated chip and etc.
This research is focus on developed the DNA amplification and detection devices by using MEMS technology. A rapid polymerase chain reaction (PCR) device and label-free microcantilever array detection chip were provided in the research.
The PCR of rotation mechanism was presented to amplify the DNA the sample when the rotation mechanism rapidly passed through the three different temperature zones, and thermal cycle time of PCR process was saved. In design and fabrication processes, CFD-ACE+ software was utilized to simulate the temperature distribution. Metal deposition and KOH wet etching technologies were used to fabricate the thin-film heater and DNA chamber. Finally, the E. Coli as the test sample was to be performed for the 100bp DNA segment amplification.
The microcantilever array structure was designed and fabricated to be as the detection chip. The SU-8 photoresist was proposed to make the mold in this research, and it was different from traditional surface micromachining and bulk micromachining. The microcantilever arry was then integrated into channel chip. For the detection method, both of fluorescence and optical system detection ways were performed to detect E. Coli DNA.

ACKNOWLEDGMENTS
ENGLISH ABSTRACT
CHINESE ABSTRACT
TABLE OF CONTENTS
LIST OF FIGURES
LIST OF TABLES
CHAPTER
Ⅰ.INTRODUCTION
1.1 Motivation
1.2 Research Goal
1.3 Thesis Organization
Ⅱ. LITERATURE REVIEW
2.1 Biochip
2.1.1 Microarray Chip
2.1.2 Microfluidics Chip
2.2 Polymerase Reaction Chain Review
2.2.1 PCR Concept
2.2.2 Continuous Flow PCR Chip
2.2.3 Micro Chamber PCR Chip
2.2.4 Conclusion
2.3 DNA Detection Review
2.3.1 Microcantilever Beam Detection Concept
2.3.2 Measurement Methods
2.3.3 Optical Read-out
2.3.4 Piezo-Resistive Read-Out
2.3.5 Fabrication Methods
2.3.6 Conclusion
Ⅲ. DESIGN OF DNA AMPLICICATION AND DETECTION
DEVICES
3.1 The Microchannel Polymerase Chain Reaction Chip
3.1.1 Research Goal
3.1.2 Design Concept
3.1.3 Thin-Film Heater Theory
3.1.4 Temperature Simulation in Microchannel
3.1.5 Conclusion
3.2 The Polymerase Chain Reaction Platform
3.2.1 Research Goal
3.2.2 Design Concept
3.2.3 Temperature Simulation of DNA Chamber
3.2.4 Conclusion
3.3 Microcantilever Array For DNA Detection
3.3.1 Research Goal
3.3.2 Design Concept
3.3.3 Thin-Film Stress Theory
3.3.4 Microcantilever Array Design
3.3.5 Mask Layout
3.3.6 Conclusion
3.4 Channel Chip Design
3.4.1 Research Goal
3.4.2 Design Concept
3.4.3 Simulation in Channel Chip
3.4.4 Conclusion
Ⅳ. FABRICATION OF DNA AMPLIFICATION DEVICES
4.1 Fabrication of Microchannel PCR Chip
4.1.1 Thin-Film Heater
4.1.2 Microchannel Fabrication
4.1.3 PCR Chip Bonding
4.1.4 Experiment and Results
4.1.5 Conclusion
4.2 Fabrication of PCR Platform
4.2.1 Thin-Film Heater and Temperature Control
4.2.2 Fabrication of Microchamber
4.2.3 PCR Platform Set Up
4.2.4 Experiment and Result
4.2.5 Conclusion
Ⅴ. FABRICATION OF MICROCANTILEVER DNA CHIP
5.1 Material Choice
5.1.1 Eposy-Based SU-8 Photoresist
5.1.2 PDMS (polydimethylsiloxane)
5.2 Fabrication of Microcantilever Array
5.2.1 Fabrication Method and Goal
5.2.2 Fabrication of SU-8 Master
5.2.3 Fabrication of PDMS Master
5.2.4 Procedure of Microcantilever Molding
5.3 Fabrication of Channel Chip
5.4 Experiment Results and Discussions
5.4.1 Silicon Etching Rate
5.4.2 PDMS Reversible Sealing
5.4.3 Cross-Section of PDMS Master
5.5 Conclusion
Ⅵ. OPTICAL SYSTEM SET UP AND MEASUREMENT
6.1 Optical System Goal
6.2 Optical Components
6.2.1 Laser Introduction
6.2.2 Laser Diode
6.2.3 Diffraction Limited
6.3 Optical Mechanism Design
6.4 Cantilever Deflection Measurement Method
6.5 Optical System Set Up
6.6 Measurement Processes
6.6.1 DNA Sample
6.6.2 Fluorescent Detection
6.6.3 Optical System Measurement
6.7 Conclusion
Ⅶ. CONCLUDING REMARKS
7.1 Microchannel PCR Chip
7.2 PCR Platform
7.3 DNA Detection Chip
7.4 Future Works
REFERENCES

[1] Jing Cheng, Larry J. Kricka, “Biochip Technology”, harwood academic publishers, pp.107-108,245-246, 2001.
[2] 產業調查與技術第一四一期
[3] Caliper Technologies, Inc., http://www.calipertech.com/index.html, 11.12.2000
[4] Max-Planck-Institut, Mainz, http://www.mpip-mainz.mpg.de , 11.12.2000
[5] Daniel J. Sadler, Rajnish Changrani, Peter Roberts, Member, IEEE, Chia-Fu Chou, Frederic Zenhausern, ”Thermal Management of BioMEMS: Temperature Control for Cermic-Based PCR and DNA Detection Devices, “IEEE Transactions on Components and Packaging Technologies, vol.26, no.2, June 2003.
[6] C. F. Chou, R. Changrani, P. Roberts, D. Sadler, J. Burdon, F. Zenhausern, S. Lin, A. Mulholland, N. Swami, R. Terbrueggen, “A miniaturized cyclic PCR device—modeling and experiments, “Microelectronic Engineering 61–62, pp.921–925, 2002.
[7] Ivonne Schneegaß and Johann Michael Köhler, ”Flow-through polymerase chain reactions in chip thermocyclers, “ Reviews in Molecular Biotechnology 82, pp.101-121, 2001.
[8] Martin U. Kopp, Andrew J. de Mello, Andreas Manz, “chemical amplification: continuous-flow PCR on a chip, “Science vol.280, pp.1046-1048, 1998.
[9] Tatsuhiro Fukuba, Takatoki Yamamoto, Takeshi Naganuma, Teruo Fujii, ”Microfabricated flow-through device for DNA amplification—towards in situ gene analysis, ”Chemical Engineering Journal 101, pp.151–156, 2004.
[10] Kai Sun, Akira Yamaguchi, Yutaka Ishida, Shigeki Matsuo, Hiroaki Misawa, “A heater-integrated transparent microchannel chip for continuous-flow PCR, ”Sensors and Actuators B 84 (2002) 283–289.
[11] 科學發展, vol.349, Jan. 2002
[12] Jeong Ah Kim, Ji Youn Lee, Shimyoung Seong, Seung Hwan Cha,
Seung Hwan Lee, Jae Jeong Kim, Tai Hyun Park, “Fabrication and characterization of a PDMS-glass hybrid comtinuous-flow PCR chip, “Biochemical Engineering Journal 29, pp.91–97, 2006.
[13] Pierre J. Obeid, Theodore K and Christopoulos, “Continuous-flow DNA and RNA amplification chip combined with laser-induced fluorescence detection, “Analytica Chimica Acta 494, pp.1–9, 2003.
[14] Nathaniel C. Cady et al, “Real-time PCR detection of Listeria monocytogenes using an integrated microfluidics platform, “Sensors and Actuators B 107 (2005) 332-341.
[15]全華科技圖書股份有限公司，林螢生編著,”光電子學-原理、元件與應用,”2002.
[16] 全華科技圖書股份有限公司，孫慶成 編著,”光電概論,”2001
[17] 合記圖書出版社,李權益 編著,”分子生物學,”2002.
[18] Holger Becker and Ulf Heim, “Hot embossing as a method for the fabrication of polymer high aspect ratio structures, “Sensors and Actuators A 83, pp.130-135, 2000.
[19] Tatsuhiro Fukuba, Takeshi Naganuma and Teruo Fujii, “Microfabricated flow-through PCR device for underwater microbiological study, “IEEE pp.101-105, 2002.
[20] Byung-Ho Jo, Linda M. Van Lerberghe, Kathleen M. Motsegood, and David J. Beebe, Member, IEEE., “Three-Dimensional Micro-Channel Fabrication in Polydimethylsiloxane (PDMS) Elastomer, “Journal of microelectromechanical systems, vol.9, no.1, pp.76-81, 2000.
[21] J. Felbel, I. Bieber and J. Pipper, J.M. Köhler, “Investigations on the compatibility of chemically oxidized silicon(SiOx)-surfaces for applications towards chip-based polymerase chain reaction, “Chemical Engineering Journal 101 pp.333–338, 2004.
[22] J. El-Ali, I.R. Perch-Nielsen, C.R. Poulsen, D.D. Bang, P. Telleman, A. Wolff, “Simulation and experimental validation of a SU-8 based PCR thermocycler chip with integrated heaters and temperature sensor, “Sensors and Actuators A 110, pp.3–10, 2004.
[23] Xiaomei Yu, Dacheng Zhang, Ting Li, Lin Hao, Xiuhan Li, “3-D microarrays biochip for DNA amplification in polydimethylsiloxane (PDMS) elastomer, “Sensors and Actuators A 108, pp.103–107, 2003.
[24] Yu-Cheng Lin, Ming-Yuan Huang, Kung-Chia Young, Ting-Tsung Chang, Ching-Yi Wu, “A rapid micro-polymerase chain reaction system for hepatitis C virus amplification, “Sensors and Actuators B 71, pp.2-8, 2000.
[25] Ivan Erill, Susana Campoy, Nadina Erill, Jordi Barbé, Jordi Aguiló, “Biochemical analysis and optimization of inhibition and adsorption phenomena in glass–silicon PCR-chips, “Sensors and Actuators B 96, pp.685–692, 2003.
[26] Lin Chen, Jicun Ren, Rui Bi, Di Chen, ”Ultraviolet sealing and poly(dimethylacrylamid) modification for poly(dimethylsiloxane)/glass microchips, ” Electrophoresis 25, pp.914-921, 2004.
[27] Zhan Zhao, Zheng Cui, Dafu Cui, Shanhong Xia, “Monolithically integrated PCR biochip for DNA amplification, “Sensors and Actuators A 108, pp.162–167, 2003.
[28] C. G. J., Evans, A. G. R., Brunnschweiler, A., Ensell, G. J., Leslie, D. L. and Lee, M. A., “Closed Chamber PCR-chips for DNA amplification, Eengineering Science and Education journal, pp.259-264, 2000.
[29] J. W. Hong, T Fujii, M Seki, T Yamamoto, “PDMS-glass hybrid microchip for gene amplification, “IEEE-EMBS Special Conference on Microtechnologies in Medicine & Biology, pp.407-410, 2000.
[30] Yan Weiping, Du Liqunb, Wang Jing, Ma Lingzhi, Zhu Jianbo, “Simulation and experimental study of PCR chip based on silicon,” Sensors and Actuators B 108, pp.695–699, 2005.
[31] Nathaniel C. Cady, Scott Stelick and Carl A. Batt, “Nucleic acid purification using microfabricated silicon structures, “Biosensors and Bioelectronics 19, pp.59-66, 2003.
[32] Keyue Shen, Xiaofang Chen, Min Guob, Jing Cheng,, “A microchip-based PCR device using flexible printed circuit technology, “Sensors and Actuators B 105, pp.251–258, 2005.
[33] J. Fritz, M. K. Baller, H. P. Lang, H. Rothuizen, P. Vettiger, E. Meyer, H.-J. Güntherodt, Ch. Gerber, J. K. Gimzewski, “Translating Biomolecular Recongnition into Nanomechanics, ”Science, vol. 288, pp.316-318, 2000.
[34] Rüdiger Berger, Emmanuel Delamarche, Hans Peter Lang, Christoph Gerber, James K. Gimzewski, Ernst Meyer, Hans-Joachim Gü ntherodt, “Surface stress in the self-assembly of alkanethiols on gold, “Science, vol.276, pp.2021-2024, 1997.
[35] M. Calleja, M. Nordström, M. Álvarez, J. Tamayo, L.M. Lechuga, A. Boisen, “Highly sensitive polymer based cantilever sensors for DNA detection, “Ultramicroscopy 105, pp.215–222, 2005.
[36] Roberto Raiteri, Massimo Grattarola, Hans Jürgen Butt and Petr Skládal, “Micromechanical cantilever-based biosensors, “Sensors and Actuators B 79, pp.115–126, 2001.
[37] L.G. Carrascosa, M. Moreno, M. Álvarez, L.M. Lechuga, “Nanomechanical biosensors: a new sensing tool, “Trends in Analytical Chemistry, vol. 25, no.3, pp.196-206, 2006.
[38] Alexander Bietsch, Jiayun Zhang1, Martin Hegner1, Hans Peter Lang, and Christoph Gerber, “Rapid functionalization of cantilever array sensors by inkjet printing, “Nanotechnology 15, pp.873–880, 2004.
[39]http://otir.nci.nih.gov/index.html
[40] http://nanocenter.nchu.edu.tw/afm/afm_1.html
[41] Daisuke Saya, Kimitake Fukushima, Hiroshi Toshiyoshi, Gen Hashiguchi, Hiroyuki Fujita, and Hideki Kawakatsu, “Fabrication of single-crystal Si cantilever array, “Sensors and Actuators A, vol. 95, pp.281-287, 2002.
[42] Jianhong Pei, Fang Tian, and Thomas Thundat, “Glucose Biosensor Based on the Microcantilever, “Anal. Chem. 76, pp.292-297, 2004.
[43] Kwang-Ho Na, Yong-Sang Kim and C.J. Kang, “Fabrication of piezoresistive microcantilever using surface micromachining technique for biosensors, “Ultramicroscopy 105, pp. 223–227, 2005.
[44] J. C. McDonald and D. C. Duffy,” Fabrication of microfludic systems in poly(dimethylsiloxane),” Electrophoresis 21, pp.27-40, 2000.
[45] Francois Huber, Martin Hegner, Christoph Gerber, Hans-Joachim Güntherodt, Hans Peter Lang, “Label free analysis of transcription factors using microcantilever arrays, “Biosensors and Bioelectronics 21, pp.1599–1605, 2006.
[46] Min Yue, Henry Lin, Daniel E. Dedrick, Srinath Satyanarayana, Arunava Majumdar, Fellow, ASME, Aditya S. Bedekar, Jerry W. Jenkins, and Shivshankar Sundaram, “A 2-D Microcantilever Array for Multiplexed Biomolecular Analysis, “Journal of Microelevtromechanical System, vol. 13, no.2, pp.290-299, 2004.
[47] MarÀlvarez and Javier Tamayo, “Optical sequential readout of microcantilever arrays for biological detection, “Sensors and Actuators B 106, pp.687–69, 2005.
[48] HANS-JüRGEN BUTT, “A Sensitive Method to Measure Changes in the Surface Stress of Solids, “Journal of Colloid and Interface Seience 180, pp.251–260, 1996.
[49] Roberto Raiteri, Massimo Grattarola, Hans-Jürgen Butt, Petr Skládal, “Micromechanical cantilever-based biosensors, “Sensors and Actuators B 79, pp.115-126, 2001.
[50] Cagri A. Savran, Andrew W. Sparks, Joachim Sihler, Jian Li, Wan-Chen Wu, Dean E. Berlin, Thomas P. Burg, Student Member, IEEE, Jürgen Fritz, Martin A. Schmidt, Senior Member, IEEE, and Scott R. Manalis, “Fabrication and Characterization of a Micromechanical Sensor for Differential Detection of Nanoscale Motions, “Journal of microelectromechanical systems, vol. 11, no. 6, pp.703-708, 2002.
[51] A. Johansson, M. Calleja, P.A. Rasmussen, A. Boisen, “SU-8 cantilever sensor system with integrated readout, “Sensors and Actuators A 123–124, pp.111–115, 2005.
[52] Rodolphe Marie, Henriette Jensenius, Jacob Thaysen, Claus B. Christensen,
Anja Boisen, “Adsorption kinetics and mechanical properties of thiol-modi.ed DNA-oligos on gold investigated by microcantilever sensors, “Ultramicroscopy 91, pp.29–36, 2002.
[53] J H He, S Q Sun, Y F Li, J S Ye, J M Kong, Y L Cai, C.W.H. Li, T M Lim, W C Hui, “Micro-Cantilever Resonance Sensor for Biomolecular Detection by Using Self-Assembly Nano-Particles, “Proc. of SPIE, vol. 6112, 61120S, pp.61120S-1- 61120S-11, 2005.
[54] J.H.T. Ransley, M. Watari, D. Sukumaran, R.A. McKendry, A.A. Seshia, ”SU8 bio-chemical sensor microarrays, “Microelectronic Engineering 83, pp.1621–1625, 2006.
[55] Maria Nordstrm, Montserrat Calleja and Anja Boisen, “ Polymeric micro-channel-based functionalisation system for micro-cantilevers, “Ultramicroscopy 105, pp.281–286, 2005.
[56] Srinath Satyanarayana, Daniel T. McCormick andArun Majumdar, “Parylene micro membrane capacitive sensor array for chemical and biological sensing, “Sensors and Actuators B 115, pp.494–502, 2006.
[57] Mingqiang Yi, Ki-Hun Jeong and Luke P. Lee, “Theoretical and experimental study towards a nanogap dielectric biosensor, “Biosensors and Bioelectronics 20, pp.1320–1326, 2005.
[58] Takao Masuda, Akira Yamaguchi, Masayuki Hayashid, Fumika Asari-Oi,
Shigeki Matsuo, Hiroaki Misawa, “Visualization of DNA hybridization on gold thin film by utilizing the resistance effect of DNA monolayer, “Sensors and Actuators B 105, pp.556–561, 2005.
[59] Yeolho Lee, Geunbae Lim and Wonkyu Moon, “A self-excited micro cantilever biosensor actuated by PZT using the mass micro balancing technique, “Sensors and Actuators A 130–131, pp.105–110, 2006.
[60] Robert L. Gunter, Rosalie Zhine, William G. Delinger, Kevin Manygoats, Ara Kooser, and Timothy L. Porter, “Investigation of DNA Sensing Using Piezoresistive Microcantilever Probes, “IEEE Sensors Journal, vol.4, no.4, pp.430-433, 2004.
[61] Guanghua Wu, Haifeng Ji, Karolyn Hansen, Thomas Thundat, Ram Datar, Richard Cote, Michael F. Hagan, Arup K. Chakraborty, and Arunava Majumdar, “Origin of nanomechanical cantilever motion generated from biomolecular interadtions, “PNAS vol.98, no.4, pp.1560-1564, 2001.
[62] L.M. Lechuga, J. Tamayo, M. Álvarez, L.G. Carrascosa, A. Yufera, R. Doldán, E. Peralías, A. Rueda, J.A. Plaza, K. Zinoviev, C Domínguez, A. Zaballos, M. Moreno, C. Martínez-A, D. Wenn, N. Harris, C. Bringer, V. Bardinal, T. Camps, C. Vergnenégre, C. Fontaine, V. Díaz e, A. Bernad, “A highly sensitive microsystem based on nanomechanical biosensors for genomics applications, “Sensors and Actuators B 118, pp.2–10, 2006.
[63] T. Sulchek, R. Hsich, S. C. Minne, and C. R. Quate, “interdigital cantilever as a biological sensor, “IEEE-NANO Nanotechnology for sensing, pp.562-566, 2001.
[64] Yoshinori Yokoyama, Munehisa Takeda, Toshiyuki Umemoto, and Tetsurou Ogushi, “Thermal micro pumps for a loop-type micro channel, “Sensors and Actuators A 111, pp.123–128, 2004.
[65] V. Hessel, H. Lowe, and F. Schonfeld, “Micromixers—a reviewon passive and active mixing principles, “Chemical Engineering Science 60, pp.2479-2501, 2005.
[66] Beebe D.J., Adrian R.J., Olsen M.G., Stremler M.A., Aref H., Jo B.-H., “ Passive mixing in microchannels: Fabrication and flow experiments, “Mecanique Industries, vol.2, pp.343-348, 2001.

[67] Hironobu Sato., Takayuki Kakinuma, Jeung Sang Go, Shuichi Shoji, “In-channel 3-D micromesh structures using maskless multi-angle exposures and their microfilter application, “Sensors and Actuators A 111, pp.87-92, 2004.
[68] Holger Becker and Ulf Heim, ”Hot embossing as a method for the fabrication of polymer high aspect ratio structures,”Sensors and Actuators 83, pp.130–135, 2000.
[69] Holger Becker and Laurie E. Locasciob, ”Polymer microfluidic devices, ”Talanta 56, pp.267–287, 2002.
[70] Jessamine Ng Lee, Cheolmin Park and George M. Whitesides, ”Solvent Compatibility of Poly(dimethylsiloxane)-Based Microfluidic Devices, ”Anal. Chem. 75, pp.6544-6554, 2003.
[71] Jessamine M. K. Ng, Irina Gitlin, Abraham D. Stroock, George M. Whitesides, “Components for integrated poly-dimethylsiloxane microfluidic systems, ”Eeletrophoresis 23, pp.3461-3473, 2002.
[72] http://rdo.ntu.edu.tw/research/sslu/sslu.htm
[73] http://en.wikipedia.org/wiki/Polydimethylsiloxane
[74] J C Lötters, W Olthuis, P H Veltink and P Bergveld, “The mechanical properties of the rubber elastic polymer polydimethylsiloxane for sensor applications, “J. Micromechanics and Microengineering 7 pp.145-147, 1997.
[75]http://www.microchem.com
[76] M.Sc. Thesis, “Fabrication of Cantilever Chip with Complementary Micro Channel System in SU-8 for Biochemical Detection,” Lektor Carl-Erik Magnusson, Fysicum, Lund University January 2004.
[77] http://www.geocities.com/guerinlj.
[78] Hai-Feng Ji and Thomas Thundat, “In situ detection of calcium ions with chemically modified microcantilevers, “Biosensors & Bioelectronics 17, pp.337–343, 2002.