臺灣博碩士論文加值系統

本研究利用微機電技術製作三極式電化學微感測電極監測生理參數Glucose、Lactate，並針對微小化參考電極需具穩定性、可再現性、可靠性之需求，以及考量植入式感測器可能面臨之問題，提出藉由覆蓋於感測器上之保護膜(Protective membrane)來改善，期望除了能提高感測器之生物相容性或耐久性外，進而可同時提高微小化參考電極的穩定性。此外，為了將檢測系統即電化學感測系統之架構簡化與微小化，本研究也設計出單電源(+3V)定電壓之電化學感測電路，達到最終微小化之目的。
觀察不同保護膜材料對參考電極之穩定性探討，由結果顯示Polyurethane(PU)保護膜確實能改善參考電極之穩定性，因此後續實驗以Polyurethane(PU)保護膜為主，觀察其對酵素電極之感測影響，結果發現具Polyurethane(PU)保護膜之Glucose biosensor或Lactate biosensor並不會影響其感測能力，且動物實驗中得知植入數天後具Polyurethane(PU)保護膜之Glucose biosensor或Lactate biosensor同樣不會影響其感測能力！而觀察植入後之感測器電極表面，發現所有感測器植入數天後電極表面之Polyurethane(PU)保護膜結構皆無破壞，仍具保護功能！

In our study, preparation of the three-electrode biosensors used the MEMS technology. The three-electrode biosensors measured glucose and lactate using glucose oxidase (GOD) and lactate dehydrogenase (LDH). Biomedical and implantable sensor applications require stable, reproducible, reversible, and reliable miniaturized reference electrode , made of biocompatible materials. So the present study reports the application of protective membrane on the device, which exists both biocompatibility and permselectivity, and acts as a surface coating material for the outer layer of the Bio-sensor. For miniaturization of sensing system, we design a single-supply circuit.
Thus, our study used difference protective membrane to assure the long-term stability of the Ag/AgCl reference electrode. From those results, we can know that reference electrode with polyurethane protective membrane is more stable than the one without protective membrane. In vivo performance, the sensor with polyurethane protective membrane can also work when implanted subcutaneously in a rat.

口試委員會審定書 i
誌謝 ii
中文摘要 iii
英文摘要 iv
目錄 v
圖目錄 vii
表目錄 xi


第一章 緒論 1
1-1 前言 1
1-2 研究背景 2
1-2-1文獻回顧 2
1-2-2生理參數之選擇 7
1-3 研究動機與目的 9
1-4 論文之架構 11

第二章 實驗理論基礎 12
2-1 生物感測器 12
2-2 電化學生物感測器 17
2-2-1 何謂電化學 17
2-2-2 電化學反應種類 18
2-2-3 電極與溶液介面關係 21
2-2-4 電化學分析方法 22

第三章 實驗方法 25
3-1 三極式電化學微感測電極之研製 25
3-1-1 三極式電化學微感測電極之規格 25
3-1-2 微機電技術製作三極式電極之流程與方法 26
3-1-3 微機電技術製作三極式電極之製程技術 28
3-1-3-1 晶片清洗 28
3-1-3-2 光微影術 28
3-1-3-3 物理薄膜沉積 32
3-1-3-4 光阻剝離法 33
3-1-3-5 電鍍RE 34
3-2 電化學感測電極之修飾與感測 35
3-2-1 GOD、LDH感測機制 35
3-2-2 Polymer包埋固定酵素 37
3-2-3 保護膜(Protective membrane)之製作 39
3-2-3-1 Nafion保護膜之製作 39
3-2-3-2 Polyurethane保護膜之製作 41
3-2-4 實驗藥品與儀器 44
3-3 量測平台微小化之建立 47
3-3-1 電化學電路設計概念 48
3-3-2 單電源定電壓電化學感測電路設計 52

第四章 動物實驗測試 54
4-1 植入生物體之實驗步驟(Implantation) 54
4-2 In vivo量測與生物相容性評估 60

第五章 實驗結果與討論 61
5-1 三極式電化學微感測電極之研製 61
5-1-1 三極式電化學微感測電極完成實體 61
5-1-2 三極式電化學微感測電極功能性驗證 64
5-2 電化學微感測電極之之修飾與感測 66
5-2-1 保護膜對參考電極( Reference Electrode)之穩定性探討 66
5-2-2 Polyurethane(PU)對酵素電極之測試 69
5-2-3 Polyurethane(PU)對酵素電極之穩定性探討 72
5-3 量測平台微小化之建立 73
5-4 動物實驗之測試 77
5-4-1功能性測試 77
5-4-1-1 葡萄糖氧化酶(Glucose Oxidase,GOD) 功能測試 79
5-4-1-2 乳酸脫氫酵素 ( L-Lactic Dehydrogenase,LDH) 功能測試 81
5-4-2 生物相容性評估 84

第六章 結論與未來展望 92
6-1 結論 92
6-2 未來展望 93

參考文獻 95

[1] Heidi E. Koschwanez, William M. Reichert , ”In vitro, in vivo and post explantation testing of glucose-detecting biosensors: Current methods and recommendations” Biomaterials, 28 ,2007, 3687–3703
[2] Koschinsky, T., Heinemann, L., “ Sensors for glucose monitoring: technical and clinical aspects.” Diabetes/Metab. Res. Rev. 17 (2), 2001, 113–123.
[3] Wisniewski, N., Moussy, F., Reichert, W.M., ”Characterization of implantable biosensor membrane fouling” Fresen. J. Anal. Chem. ,366, 2000, 611–621.
[4] Chien-Yuan Chen, Kazuhiko Ishihara, Nobuo Nakabayashi, Eiichi Tamiya, and Isao Karube, “Multifunctional Biocompatible Membrane and Its Application to Fabricate A Miniaturized Glucose Sensor with Potential for Use In Vivo” Journal of Biomedical Microdevices 1,2, 1999, 155-166,
[5] W.J. Sung et al., ”Biocompatibility and interference eliminating property of pullulan acetate(PA)/polyethylene glycol(PEG)/heparin membrane for the outer layer of an amperometric glucose sensor” Sensors and Actuators B ,99 ,2004, 393–398
[6] Bazhang Yu et al., ”COIL-TYPE IMPLANTABLE GLUCOSE BIOSENSOR WITH EXCESS ENZYME LOADING” Frontiers in Bioscience ,10 ,2005, 512-520
[7] B. Yu et al., “A long-term flexible minimally-invasive implantable glucose biosensor based on an epoxy-enhanced polyurethane membrane” Biosensors and Bioelectronics , 21,2006, 2275–2282
[8] www.sbu.ac.uk/biology/enztech/immethod.html
[9] Pablo A et al. ”Glucose Amperometric Biosensor Based on the Co-immobilization of Glucose Oxidase (GOx)and Ferrocene in Poly(Polypyrrole) Generated from Ethanol / Water Mixtures” J. Braz. Chem. Soc.,12,No.6, 2001, 729-733

[10] Almira Ramanaviciene, Asta Kausaite, Stasys Tautkus and Arunas Ramanavicius, “ Biocompatibility of polypyrrole particles: an in-vivo study in mice” Journal of Pharmacy and Pharmacology , 59, 2007,311-315
[11] Won Jun Sung, You Han Bae, ,”Glucose oxidase, lactate oxidase, and galactose oxidase enzyme electrode based on polypyrrole with polyanion/PEG/enzyme conjugate dopant, “ Sensors and Actuators B ,114 ,2006, 164–169
[12] Cosnier, S., “Biomolecule immobilization on electrode surface by entrapment or attachment to electrochemically polymerized films.” Biosens. Bioelectron. 14, 1999, 443–456
[13] 國際糖尿病協會http://www.cvd.idf.org/
[14] Berruet, R., Vinard, E., Calle, A., Tighzert, H.L., Chabert, B., Magloire, H., Eloy, R. , “Mechanical properties and biocompatibility of two polyepoxy matrices: DGEBA-DDM and DGEBA-IPD.” Biomaterials 8 (3), 1987, 162–171.
[15] Nakamura, M., “Biocompatibility of epoxy-treated bioprosthetic heart valves. “ Yokohama Igaku , 48 (2), 1997, 165–174.
[16] Geckeler, K., Wacker, R., Martini, F., Hack, A., Aicher, W., “Enhanced biocompatibility for SAOS-2 osteosarcoma cells by surface coating with hydrophobic epoxy resins” , Cell Physiol. Biochem. 13 (3), 2003,155–164.
[17] Xinxia Cai, Chunxiu Liu, Hongmin Liu, Zonghui Guo, Juntao Liu, “Optimization of Electrochemical Total Creatine Kinase Biosensors Based on Three- Au- Electrode ” ,Electroanalysis ,19, No. 16, 2007, 1689 – 1694
[18] Chun-Xiu Liu , Li-Ying Jiang, Hui Wang , Zong-Hui Guo, Xin-Xia Cai, “A novel disposable amperometric biosensor based on trienzyme electrode for the determination of total creatine kinase” ,Sensors and Actuators B, 122,2007, 295–300

[19] L. Piras, S. Reho, “ Colloidal gold based electrochemical immunoassays for the diagnosis of acute myocardial infarction” , Sensors and Actuators B, 111–112 ,2005, 450–454
[20] Sungho Ko, Bumjun Kim, Seong-Sik Jo , Se Young Oh, Je-Kyun Park, “ Electrochemical detection of cardiac troponin I using a microchip with the surface-functionalized poly(dimethylsiloxane) channel” , Biosensors and Bioelectronics ,23 ,2007, 51–59
[21] Yu-Chen Tsai , Siao-Yun Chen, Hong-Wei Liaw , “Immobilization of lactate dehydrogenase within multiwalled carbon nanotube-chitosan nanocomposite for application to lactate biosensors “ ,Sensors and Actuators B, 125 , 2007, 474–481
[22] Arnaldo C. Pereira , Marina R. Aguiar, Alexandre Kisner , Denise V. Macedo, Lauro T. Kubota, “Amperometric biosensor for lactate based on lactate dehydrogenase and Meldola Blue coimmobilized on multi-wall carbon-nanotube”, Sensors and Actuators B ,124 ,2007, 269–276
[23] http://topvet.topet.net/clinic_38.htm
[24] 巫鴻章、林畢修平, ”高效率電化學式農藥生物微感測器開發”, 農業生技產業季刊, 8, 2006, 25-33
[25] 李坤易, ”高感度葡萄糖生物感測器之研究”, 國立雲林科技大學化學工程系碩士論文, 2006,6
[26] 廖晟偉, ”微電化學電極陣列感測平台於血糖與膽固醇量測之研發”, 台灣大學碩士學位論文,2005,6.
[27] Allen J.Bard , Larry R.Faulkner, “ELECTROCHEMICAL METHODS Fundamentals and Applications 2nd” , Fig 12.1.1
[28] 李明峰, ”比咯高分子於微型生醫感測元件之表面改質與細胞生理應用之探討”, 台灣大學碩士學位論文,2004,6.
[29] A. ul Haque, M. Rokkam, A.R. De Carlo , S.T. Wereley , S.J. Roux, P.P. Irazoqui , D.M. Porterfield , “A MEMS fabricated cell electrophysiology biochip for in silico calcium measurements ” ,Sensors and Actuators B , 123 , 2007, 391–399
[30] S. Koide , N. Ito, I. Karube , ”Development of a micro-planar amperometric bile acid biosensor for urinalysis” ,Biosensors and Bioelectronics, 22 , 2007, 2079–2085
[31] Chun-Xiu Liu , Li-Ying Jiang , Hui Wang , Zong-Hui Guo, Xin-Xia Cai , ”A novel disposable amperometric biosensor based on trienzyme electrode for the determination of total creatine kinase” ,Sensors and Actuators B, 122,2007, 295–300
[32] Hui Xu, Guang Li, Jieying Wu, You Wang, and Jun Liu, ”A glucose oxidase sensor based on screen-printed carbon electrodes modified by polypyrrole ”, Engineering in Medicine and Biology 27th Annual Conference Shanghai, China, September 1-4, 2005, 1917-1920
[33] Jesus De; Robert Weiss, “Conductive polymer blends prepared by in situ polymerization of pyrrole: a review” , Polymer engineering and science, 37,1997, 1936-1943.
[34] 周澤川, “定電流聚合導電性高分子法製備葡萄糖生物感測器之研究” 成功大學博士學位論文,2003,6
[35] S. Ahmed, C. Dack, G. Farace , G. Rigby , P. Vadgama, “Tissue implanted glucose needle electrodes: early sensor stabilisation and achievement of tissue-blood correlation during the run in period” , Analytica Chimica Acta ,537 ,2005,153–161
[36] Ran-A Jeong , Jae Youn Hwang, Segyeong Joo, Taek Dong Chung, Sejin Park, Sun Kil Kang , Won-Yong Lee , Hee Chan Kim , “In vivo calibration of the subcutaneous amperometric glucose sensors using a non-enzyme electrode”, Biosensors and Bioelectronics ,19 ,2003, 313-319

[37] G.A.M. Mersal , M. Khodari , U. Bilitewski, “Optimisation of the composition of a screen-printed acrylate polymer enzyme layer with respect to an improved selectivity and stability of enzyme electrodes” , Biosensors and Bioelectronics ,20, 2004, 305–314
[38] Allen J.Bard , Larry R.Faulkner , “ELECTROCHEMICAL METHODS Fundamentals and Applications 2nd ” , Fig 15.4.5 ,Electronics of Potentiostat
[39] Gabriela Voskerician, Rebecca S. Shawgo, P. Anne Hiltner, James M. Anderson, Michael J. Cima, and Robert Langer, ” In Vivo Inflammatory and Wound Healing Effects of Gold Electrode Voltammetry for MEMS Micro-Reservoir Drug Delivery Device”, IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, vol. 51, No. 4, APRIL 2004
[40] Madalina Ciobanu, Jeremy P. Wilburn, Nicholas I. Buss, Pouvan Ditavong, Daniel A. Lowy, “Miniaturized Reference Electrodes Based on Ag/AgiX Internal Reference Elements. I. Manufacturing and Performance “, Electroanalysis, 14, No. 14, 2002, 989-997
[41] Christophe A. Marquette , Lo¨ıc J. Blum, “Review State of the art and recent advances in immunoanalytical systems” ,Biosensors and Bioelectronics, 21,2006,1424–1433
[42] Olivier Lazcka , F. Javier Del Campo, F. Xavier Mu˜noz, “Review Pathogen detection: A perspective of traditional methods and biosensors”,Biosensors and Bioelectronics ,22 ,2007 ,1205–1217