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研究生： 錢培宜
Pei-Yi Chien
論文名稱： 碳六十脂肪酵素/三酸甘油酯壓電晶體感測器研製與應用
Preparation and Application of Piezoelectric Crystal Sensor for Triglyceride Based on C60-Lipase Enzyme
指導教授： 施正雄
Shih, Jeng-Shong
學位類別： 碩士
Master
系所名稱： 化學系
Department of Chemistry
論文出版年： 2007
畢業學年度： 95
語文別： 中文
論文頁數： 121
中文關鍵詞： 三酸甘油酯 、脂肪酵素 、碳六十 、壓電晶體
論文種類： 學術論文
相關次數： 點閱：114  下載：10 
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    • 碳六十脂肪酵素/三酸甘油酯壓電晶體感測器研製與應用

    摘要

    本研究合成碳六十-脂肪分解酵素(C60-lipase)，並應用在三酸甘油酯壓電晶體生物感測器。首先，利用塗佈碳六十的石英壓電晶體感測器作為研究碳六十與脂肪分解酵素間作用力，結果顯示酵素與碳六十存在不可逆的化學性吸附現象。且研究顯示碳六十與脂肪分解酵素間作用的最適溫度為32 ℃，最適pH值為7。
    IR光譜圖利用來鑑定合成的不溶性碳六十-脂肪分解酵素(C60-lipase)。然後，使用多孔性的二氧化矽材質濾片固定化碳六十-脂肪分解酵素，這碳六十-脂肪分解酵素濾片顯示可用來催化水解三酸甘油酯溶液，造成pH值的下降。
    本研究組裝一塗佈碳六十/大環胺醚的石英壓電晶體感測器用來吸附三酸甘油酯溶液經碳六十-脂肪分解酵素催化後的產物-脂肪酸，吸附所引起的負載重量增加會造成石英晶體振盪頻率的下降，並由電腦螢幕紀錄整個催化反應的過程。本研究對三乙酸甘油酯、三丁酸甘油酯及混合三酸甘油酯溶液進行偵測，分別探討了濃度、pH值效應及人體常見生物干擾物存在下的催化反應。結果顯示，此系統對三酸甘油酯的偵測具有良好的濃度線性關係及偵測極限。三乙酸甘油酯、三丁酸甘油酯及混合三酸甘油酯溶液其偵測極限依次為13.08 mg/dL、38.32 mg/dL、6.21 mg/dL皆低於人體內三酸甘油酯正常平均值50~150 mg/dL。同時，此利用C60-lipase而塗佈C60-cryptand[2.2]之生化感測器對於三乙酸甘油酯的偵測具有良好的再現性，RSD值為1.7 % (n= 5)。

    Preparation and Application of Piezoelectric Crystal Sensor for Triglyceride Based on C60-Lipase Enzyme

    Abstract

    Immobilized fullerence C60-lipase was synthesized and applied in a piezoelectric crystal biosensor for triglycerides. Firstly, The C60 coated piezoelectric quartz crystal sensor was employed to study the interaction between C60 and lipase. It seemed to indicate that the lipase could be adsorbed on C60 by chemisorption. The optimum temperature of 32 ℃ and the optimum pH of 7 for the chemisorption between the C60 and lipase were found.
    The water insoluble C60-lipase product was synthesized and identified by IR spectrum. The C60-lipase coated SiO2 filter was also prepared. The C60-lipase filter exhibited the catalytic ability for the hydrolysis of the triglycerides resulting in the decrease in pH of solutions.
    The C60/cryptand[2.2] coated piezoelectric crystal biosensor was prepared for fatty acids , the product of the hydrolysis of the triglycerides catalyzed by C60-lipase. The adsorption of fatty acids on quartz crystal resulted in decrease in the oscillating frequency of quartz crystal. And the overall reaction process can be monitored and recorded by computer. The triglycerides e.g. triacetin, tributyrin and the mixed triglycerides were detected by the piezoelectric biosensor in this study. The effects of concentration, pH and the interference of the common biological species in human blood were also investigated. The piezoelectric sensor has exhibited good concentration linear relation and good detection limit for triglycerides. The detecting limits of the triacetin, tribuytrin and the mixed triglycerides were 13.08, 38.32 and 6.21 mg/dL, respectively which are all below the normal average value of the triglycerides in human body. The C60-cryptand[2.2] coated piezoelectric biosensor with C60-lipase also showed good reproducibility for reusage with RSD = 1.7 % (n = 5) for triglycerides.

    目錄 中文摘要 I 英文摘要 II 目錄 IV 圖目錄 VIII 表目錄 XIII 第一章、緒論 1 1-1 碳六十化學 1 1-1-1 碳六十的發現 1 1-1-2碳六十的基本性質 1 1-1-3碳六十的應用 6 1-2 冠狀醚及大環胺醚 9 1-2-1冠狀醚及大環胺醚簡介 9 1-2-2大環胺醚的應用 11 1-3 酵素化學 12 1-3-1酵素的發現 12 1-3-2酵素的命名 13 1-3-3酵素的特性 14 1-3-4固定化酵素(immobilized enzymes) 19 1-3-5 脂肪分解酵素( lipase ) 27 1-4脂質( lipid ) 31 1-4-1 脂質之簡介 31 1-4-2三酸甘油酯( Triacylglycerols ；Triglycerides ) 33 1-4-3三酸甘油酯的偵測 35 1-5 壓電晶體 36 1-5-1 壓電晶體之壓電性 36 1-5-2 石英振盪器 38 1-5-3 石英振盪器的線路 40 1-5-4振盪頻率的量測 42 1-5-5 石英微量天平(Quartz Crystal Microbalance, QCM) 44 1-5-6 液相石英壓電晶體的特性 48 1-5-7石英壓電晶體之應用 50 1-6研究動機 55 第二章、實驗部分 56 2-1實驗原理與目的 56 2-2藥品與器材 56 2-3石英晶片 56 2-3-1石英晶片之處理 56 2-3-2石英銀電極之表面塗佈液 57 2-3-3表面塗佈法 58 2-4石英振盪系統 59 2-5實驗裝置及步驟 60 2-5-1碳六十與酵素間作用力的研究 60 2-5-2碳六十－酵素之合成 62 2-5-3以pH測定儀檢驗碳六十－酵素的活性 63 2-5-4三酸甘油酯壓電生物感測器之研究 64 2-5-4.1 C60-cryptand[2.2]之合成 64 2-5-4.2碳六十－脂肪分解酵素濾片之製作 65 2-5-4.3三酸甘油酯壓電晶體感測系統 66 第三章、實驗結果與討論 67 3-1碳六十與酵素間的作用力 67 3-1-1塗佈C60/PVC石英晶體對酵素的感應 67 3-1-2碳六十與酵素間的吸附作用力 67 3-1-3C60/PVC的塗佈量效應 67 3-1-4酵素溶液的濃度效應 67 3-1-5酵素溶液的溫度效應 68 3-1-6酵素溶液的pH值效應 68 3-2碳六十固定化酵素 75 3-2-1碳六十/脂肪分解酵素的鑑定 75 3-2-2碳六十/脂肪分解酵素的活性 75 3-3碳六十/大環胺醚的鑑定 78 3-4研製三酸甘油酯壓電生化感測器 80 3-4-1三乙酸甘油酯之偵測 80 3-4-1.1 C60-Cryptand[2.2]/PVC塗佈石英晶體之感應情形 80 3-4-1.2C60-Cryptand[2.2]/PVC的塗佈量效應 80 3-4-1.3三乙酸甘油酯之濃度效應 81 3-4-1.4濾片數目(C60-Lipase量)的影響 85 3-4-1.5三乙酸甘油酯之pH值效應 85 3-4-1.6 再現性 88 3-4-1.7 其他生物分子之干擾研究 88 3-4-1.8固定化酵素濾片多次使用之活性探討 88 3-4-2三丁酸甘油酯之偵測 96 3-4-2.1塗佈C60-Cryptand[2.2]/PVC石英晶體之感測情形 96 3-4-2.2 C60-Cryptand[2.2]/PVC的塗佈量效應 96 3-4-2.3三丁酸甘油酯之濃度效應 96 3-4-2.4三丁酸甘油酯之pH值效應 100 3-4-2.5體內干擾物存在下的影響 100 3-4-2.6 不同三酸甘油酯之感應訊號 100 3-4-3三酸甘油酯混合物之偵測 107 3-4-3.1塗佈C60-Cryptand[2.2]/PVC石英晶體之感測情形 107 3-4-3.2 三酸甘油酯濃度效應 107 3-4-3.3 pH值效應 107 3-4-3.4體內干擾物存在下的影響 111 3-4-3.5濾片儲存活性 111 第四章、結論 117 參考文獻 118

    參考文獻
    1. A. Rosén, A sightseeing tour in the world of clusters—serendipity and scientificprogress. J. Mol. Graphics Modell., 2001, 19, 236-243
    2. 朱智謙, 碳六十衍生之星狀高分子的合成與分析, 國立中山大學化學研究所碩士論文, 2001
    3. 廖健良, 碳六十薄膜電性及場發射量測, 私立中原大學應用物理研究所碩士論文, 2004.
    4. http://nano.nchc.org.tw/dictionary/C60.html
    5. Kroto, H. W.; Allaf, A. W.;Balm, S. P.,C60: Buckminsterfullerence. Chem. Rev.,1991, 91, 1213.
    6. W. Chen, Z. Xu, Temperature Dependence of C60 Solubility in Different Solvent, Fullerence Science And Technology, 1998, 6(4), 695-705.
    7. Taylor, R.; Walton, D. R. M., The chemistry of fullerenes, Nature, 1993, 363, 685-693.
    8. C. F. Perderson, Cyclic polyethers and their complexs with metal salts, J. Am. Chem. Soc., 1967, 89, 7017-7036.
    9. X. X. Zhang, Reed M. Izatt *, Jerald S. Bradshaw, Krzysztof E. Krakowiak , Approaches to improvement of metal ion selectivity by cryptands, Coord. Chem. Rev., 1998, 179-189.
    10. C.H.Chen , H.W.Chang , J.S. Shih , Optical isomer piezoelectric crystal sensor for L-amino acid esters based on immobilized C60–lipase enzyme,Sen. Actuators., 2007, 123, 1025-1033
    11. 李盛、黃偉達, 構築生命-蛋白質、核酸與酶, 2002
    12. 陳國誠, 微生物酵素工程學, 1989.
    13. 鄭建業, 生物有機化學, 俊傑書局股份有限公司, 2001.
    14. 趙世彬、蔡東基、周大中、陳河吉, 生物化學, 藝軒圖書出版社,2000.
    15. Nelson Cox. Lehninger Principles of Biochemistry, WORTH. 2000
    16. 田蔚城，生物技術的發展與應用, 九州圖書文物有限公司, 1999.
    17. R. A. Messing, Immobilized Enzymes for Industrial Reactors, Academic Press, New York, 1975.
    18. 久保田 旺, 生物技術基礎, 富林出版社, 2002
    19. 相田 浩, 生物技術概論, 富林出版社, 2002.
    20. 魏麗芳, 碳六十/大環胺醚壓電晶體尿素感測器研製與應用, 國立台灣師範大學碩士論文, 1998.
    21. C.W.Chung , J.S.Shih , Preparation and application of immobilized C60-glucose oxidase enzyme in fullerene C60-coated piezoelectric quartz crystal glucose sensor, Sen. Actuators , 2001, 81, 1-8
    22. 林仁混, 生化學通論－人類疾病之生化觀, 聯經, 1987.

    23. R.Fernandez-Lafuente et al., Immobilization of lipases by selective adsorption on hydrophobic supports, Chem. Phys. Lipids., 1998, 185-197.
    24. J. Pleiss et al., Anatomy of lipase binding sites: the scissile fatty acid binding site, Chem. Phys. Lipids., 1998, 67-80
    25. Jose´ M. Manchen˜o, Marı´a A. Pernas, Marı´a J. Martı´nez Begon˜aOchoa, M. Luisa Ru´aand Juan A. Hermoso., Structural Insights into the Lipase/esterase Behavior in the Candida rugosa Lipases Family: Crystal Structure of the Lipase 2 Isoenzyme at 1.97 A° Resolution. J. Mol. Bio., 2003, 332, 1059-1069.
    26. M. Cygler, Joseph D. Schrag, Structure and conformational flexibility of Candida rugosa lipase, Bichim. Biophys. Acta., 1999, 205-214
    27. 姚少凌, Pseudomonas fluorescens 生產脂肪水解酵素固定於聚甲基丙烯醯胺以生產S-AMPA的應用, 國立清華大學碩士論文, 1999.
    28. E. N. Vulfson, Industrial applications of lipase. In: Woolley, P., Peterson, S. B., editors. Lipases－their structure, biochemistry and applications. Cambridge Univ. Press, 1994, 271-288.
    29. 徐慧雯, 臨床生物化學, 新文京開發出版股份有限公司, 2004
    30. 何敏夫, 臨床化學概論, 合記圖書出版社, 2002
    31. http://www.webhospital.org.tw/essay/essay.html?category=營養調理&pid=9627
    32. http://www.webhospital.org.tw/essay/essay.html?category=醫藥疾病&pid=13030
    33. http://www.bhp.doh.gov.tw/BHP/do/www/themeParkDocRead?themeParkDocumentId=10185&type=document&themeParkId=67
    34. R. Ravi Kumar Reddy , Anju Chadha , Enakshi Bhattacharya, Porous silicon based potentiometric triglyceride biosensor, Biosens. Bioelectron., 2001, 16, 313-317
    35. I. Basu et al., Solid state potentiometric sensor for the estimation of tributyrin and urea, Sens. Actuators B: Chem., 2005, 107, 418–423
    36. D.G. Pijanowska, A. Baraniecka, R. Wiater, G. Ginalska, J. Lobarzewski, W. Torbiez, The pH-detection of triglycerides, Sens. Actuators B: Chem., 2001 , 78, 263–266.
    37. 吳朗, 感測與轉換原理-元件與應用, 全欣資訊書局, 1992.
    38. G. Lu, A. W. Czanderna, Applications of Piezoelectric Quartz Crystal Microbalance. Elsevier Scienc .New York, 1984.
    39. T. Ikeda, Fundamentals of Piezoelectricity, Oxford Sci. Publ., 1990

    40. D. A. Buttry, M. D.Ward, Measurement of interfacial processes at electrode surfaces with the electrochemical quartz crystal microbalance, Chem. Rev., 1992, 1355-1379
    41. L. A. Geddes, L. E. Baker, Principle of Applied Biomedical Instrumentation. (3rd Ed.) John Wiley & Sons. New York, 1989, 163.
    42. S. J. Martin, G. C. Frye, A. J. Ricco, Effect of Surface Roughness on the response of Thickness-Shear Mode Resonators in Liquids, Anal Chem., 1993, 65, 2910-2922
    43. A. J. Levenson, “Cimento”, Suppl.2, Ser.1, 1967, 5, 321
    44. 紀培錦, 新電子科技雜誌, 1989, 17, 196.
    45. 湯進德, 微電子介面技術, 全華科技圖書, 1984
    46. 袁帝文; 黃柏鈞, 數位邏輯設計與分析, 全欣科技圖書, 1992
    47. 江宗達; 鍾健文編譯, IBM PC 與感測器介面的探討, 全華科技圖書, 1994
    48. J. Hlavay, G. G. Guilbault, Applications of Piezoelectric Crystal Detector in Analytic Chemistry, Anal Chem., 1977, 49(13), 1890-1898.
    49. G. Z. Sauerbrey, Z. Phys., 1959, 155, 206.
    50. S. Bruckenstein, M. Shay, Experimental Aspects of Use of the Quartz Crystal Microbalance in Solution, Electrochimica Acta., 1985, 30(10), 1295-1300.
    51. M. Thompson, A. L. Kipling, L. V. Rajakovic, Thickness-shear-mode acoustic wave sensors in the liquid phase. A review, Analyst., 1991, 116, 881-890
    52. Mandelis and Christofides, Physics, Chemistry and Technology of Solid State Gas Sensor Devices, John Wiley & Sons. New York, 1993.
    53. P. Chang, J. S. Shih, Multi-channel piezoelectric quartz crystal sensor for organic vapors. Analytica Chimica Acta., 2000, 403, 39-48.
    54. G. Barkó, J. Hlavay, Application of an artificial neural network (ANN) and piezoelectric chemical sensor array for identification of volatile organic compounds., Talanta., 1997, 44, 2237-2245.
    55. Su. Xiaodi, Covalent DNA Immobilization on Polymer-Shielded Silver-Coated Quartz Crystal MicrobalanceUsing Photobiotin-Based UV Irradiation, Biochemical and Biophysical Research Communications , 2002, 290, 962–966
    56. L. Lin, H. Q. Zhao, J. R. Li, J. A. Tang, M. X. Duan, and L. Jiang, Study on Colloidal Au-Enhanced DNA Sensing by Quartz Crystal Microbalance, Biochemical and Biophysical Research Communications , 2000, 274, 817–820
    57. J. Tao Liu,an Tang, and L. Jiang, The enhancement effect of gold nanoparticles as a surface modifier on DNA sensor sensitivity , Biochemicaland Biophysical Research Communications , 2004, 313, 3–7

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