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研究生: 張祐穎
Yu-Ying Chang
論文名稱: 葡萄糖/尿素碳六十固定化酵素選擇性電極之研製與應用
Preparation and Application of Fullerene C60 Immobilized Enzyme Electrodes for Glucose and Urea
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: 2003
畢業學年度: 91
語文別: 中文
論文頁數: 142
中文關鍵詞: 葡萄糖尿素葡萄糖氧化酵素尿素水解酵素固定化酵素選擇性電極
英文關鍵詞: glucose, glucose oxidase, urea, urease, immobilized enzyme, selective electrode
論文種類: 學術論文
相關次數: 點閱:194下載:0
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  • 中文摘要
    本研究合成固定化碳六十/葡萄糖氧化酵素(C60 -Glucose Oxidase)和固定化碳六十/尿素水解酵素(C60-Urease)並分別應用在葡萄糖及尿素選擇性C60-cryptand[2,2] / PVC電極感測系統中,用以偵測待測溶液中的葡萄糖或尿素分子。在C60-cryptand[2,2]葡萄糖薄膜電極系統中,偵測葡萄糖分子被葡萄糖氧化酵素催化為葡萄糖酸在經解離得到的氫離子,而C60-cryptand[2,2]尿素選擇性薄膜電極中,偵測水溶液中尿素分子經尿素水解酵素催化所形成的銨根離子。實驗中將分別探討碳六十固定化酵素的含量、溫度、pH值的改變及干擾因子對電極電位變化的影響。在葡萄糖選擇性薄膜電極偵測系統中,固定化C60 -Glucose Oxidase在30~35oC的溫度下,有很好的催化效果,而最適pH值則在6~7之間,且發現當膜上所含C60 -Glucose Oxidase的量增加時,催化的效果越明顯。而pH值的效應上發現,當待測溶液的pH值在6~7之間時,有最好的催化效果。此選擇性電極在葡萄糖分子濃度為10-4~10-1 M之間,有52.5mV/decade的線性電極電位反應,其穩定性及再現性都不錯。在干擾係數的測定方面,整體的選擇性係數在10-2左右,如鹼金族、鹼土族及過渡金屬。在尿素選擇性薄膜電極偵測系統中,最適pH值也在6~7之間而最佳溫度為室溫。此電極在尿素分子濃度為10-4~10-1 M之間,有51.7mV/decade的線性電極電位反應。在干擾離子方面,大部分干擾五種的選擇性係數也在10-2左右,如鹼金族、鹼土族及過渡金屬離子。

    Abstract
    Immobilized C60 -Glucose Oxidase and C60-Urease were synthesized and applied in glucose and urea C60-cryptand[2,2] / PVC electrodes to detect glucose and urea in aqueous solutions. The C60-cryptand[2,2] glucose selective membrane electrode detection system to detect the H+ ion which is the product of the gluconic acid dissociation catalyzed by C60-glucose oxidase in another PVC membrane and C60-cryptand[2,2] urea selective membrane electrode detection system to detect the NH4+ which is the production of urea hydrolysis catalyzed by C60-urease in another PVC membrane. The effects of mass of immobilized enzymes, temperature, pH value and interference ions on the potential response were investigated and discussed. For glucose selective membrane electrode detection system, it was found that the catalytic ability of immobilized C60-glucose oxidase showed an optimum temperature at 30℃~35℃ and optimum pH between 6~7, the potential response showed increase with the increased mass of immobilized C60-glucose oxidase in PVC membrane. This electrode gave a linear response 52.3mV/decade within the concentration range in 10-4~10-1 M of glucose and showed selectivity coefficients around 10-2 for most interfering ions, e.g., alkali, alkaline earth and transition metal ions. For urea selective membrane electrode detection system, the optimum is at testing solution pH value 6~7, and optimum temperature at room temperature. This urea electrode also exhibited a linear response 51.7 mV/decade within the concentration range in 10-4~10-1 M of urea and also showed selectivity coefficients around 10-2 for most interfering ions, e.g., alkali, alkaline earth and transition metal ions.

    目 錄 中文摘要 I ABSTRACT II 目 錄 IV 圖 目 錄 VII 表 目 錄 XI 第一章 緒論 1 1-1 碳六十簡介 1 1-1-1 碳六十(C60)的發現 1 1-1-2 碳六十的基本性質 2 1-1-3 碳六十的化學反應 5 1-2 冠狀醚與大環胺醚的簡介及應用 6 1-2-1 冠狀醚與大環胺醚簡介 6 1-2-2 冠狀醚錯合反應與主客化學 11 1-2-3 碳六十/大環胺醚在分離無機陰陽離子上的應用 12 1-3 酵素簡介 16 1-3-1 什麼是酵素(Enzyme) 16 1-3-2 酵素的發現 17 1-3-3 酵素的特異性或專一性(specificity) 17 1-3-3 酵素溫度效應 18 1-3-4酵素pH值效應 19 1-3-5 溶劑對酵素活性的影響 20 1-3-6 固定化酵素(Immobilized enzyme) 21 1-3-7 葡萄糖氧化酵素的介紹 29 1-3-8 尿素酵素分子構造與基質特異性 30 1-4 選擇性電極簡介 32 1-4-1 電極膜傳輸機制 32 1-4-2 選擇性電極膜之設計 37 1-4-3 陽離子選擇性電極 40 1-4-4 陰離子選擇性電極 41 1-4-5 選擇性係數Kpot的測量 42 第二章 實驗部分 46 2-1 藥品與儀器 46 2-2 實驗系統 46 2-2-1 碳六十/大環胺醚(C60 - Cryptand)之合成 47 2-2-2 碳六十/葡萄糖氧化酵素(C60 - Glucose Oxidase)之合成 47 2-2-3 電極膜的製備 49 2-2-4 實驗裝置 49 第三章 實驗與討論 53 3-1 氫離子選擇性的製備: 53 3-1-1 碳六十-大環胺醚的鑑定 53 3-1-2 電極製成的最佳條件 53 3-1-3 靜態與動態及穩定度的影響 67 3-1-4干擾離子選擇性係數之測定 72 3-2 碳六十-固定化葡萄糖氧化酵素電極 81 3-2-1 碳六十-葡萄糖氧化酵素的鑑定 81 3-2-2 C60-glucose oxidase 的活性 81 3-2-3 碳六十-固定化葡萄糖氧化酵素電極 95 3-3 銨根離子選擇性電極的製作 99 3-3-1銨根離子電極製成之最佳條件 99 3-3-2 選擇性係數的測定 109 3-4 碳六十-固定化尿素水解酵素電極 116 3-4-1碳六十-固定化尿素水解酵素的鑑定 116 3-4-2 以吸附法來製作碳六十-固定化尿素水解酵素薄膜 116 3-4-3 碳六十-固定化酵素電極 120 第四章 結論 124 參考文獻 125

    1. Kroto, H. W.; Heath, J. R.; O'Brien, S. C.; Curl, R. F., Smalley, R. E. C60:Buckminsterfullerene. Nature. 1985, 318, 162-163.
    2. Hawkins, J. M.; Meyer, A.; Lewis, T. A.; Loren, S.; Hollander, F. J. Crystal Structure of Osmylated C60 : confirmation of the soccer Ball framework. Science. 1991, 252, 312-313
    3. W. A. Scrirens, P. V. Bedworth, J. M. Tour, Purification of gram quanties of C60. A new inexpensive and facile method. J. Am. Chem. Soc. 1992, 114, 7917
    4. J. M. Hawkins, A. Meyer, T. A. Lewis, S. Loren, F. J. Hollander, Crystal structure of osmylated C60 : confirmation of the soccer ball framework. Science.1991, 252, 312
    5. Chen, W.; Xu, Z. Temperature dependence of C60 solubility in different solvent. Fullerence Science And Technology. 1998, 6 , 695
    6. Haufler, R. E.; Conceicao, J.; Chibante, L. P. F.; Chai, Y.; Byrne, N. E.; Flanagan, S.; Haley, M. M.; O’Brien, S. C.; Pan, C.; Xiao, Z.; Billups, W. E.; Ciufolini, M. A.; Smalley, R. H. Efficient production of C60 (buckminsterfullerene), C60H36, and the solvated buckide ion. J. Phys. Chem. 1990, 94(24), 8634-8636.
    7. Taylor, R.; Walton, D. R. M. The chemistry of fullerenes. Nature. 1993, 363, 685-693.
    8. Pederson, C.J. Crystalline salt complexes of macrocyclic polyethers J. Am. Chem. Soc. 1970,92,386
    9. Lehn, J. M.; Sauvage, J. P. Cryptates. XVI. [2]-Cryptates. Stability and selectivity of alkali and alkaline-earth macrobicyclic complexes J. Am. Chem. Soc. 1975, 97, 6700-6707
    10. Suzuki, K.; Yamada, H.; Sato, K.; Watanabe, K.; Hihamoto, H.; Tobe, Y.; Kobiro, K. Design and synthesis of highly selective ionophores for lithium ion based on 14-crown-4 derivatives for an ion-selective electrode Anal. Chem. 1993, 65, 3404-3410
    11. J. Jeng and J. S. Shih Sodium Ion Selective Electrode Based on Crown Ether Phosphotungstenic Acid Precipitates Analyst, 1984, 109, 641
    12. H. C. Sheu and J. S. Shih Ammonium (I) Ion Selective PVC Membrane Electrodes Based on Macrocyclic Polyethers J. Chin. Colloid & Interface Soc.1996, 19, 89-96
    13. Bijay Sarkar, Pritam Mukhopadhyay, Parimal K. Bharadwaj Laterally non-symmetric aza-cryptands: synthesis, catalysis and derivatization to new receptors Bijay Sarkar, Coordination Chemistry Reviews 2003, 236, 1~13.
    14. Pearson, R. G. Hard and soft acids and bases J. Am. Chem. Soc. 1963, 85, 3533-3539.
    15. Philip, C. J.; Jacqueline, M. G.; Stephen, F. L.; Edward, R. T. Complexation of alkali metal ions by the cryptand 4,7,13,16 -tetraoxa-1,10-diazabicyclo[8.8.5]tricosane,C22C5.A structural and equilibrium study. Inorg. Chem. 1992, 31, 3398-3404.
    16. M. Hiraoka, “Crown Compounds-their characteristics and applications”, 1982, (Kodansha, Tokyo)
    17. 麥菁菁,碩士論文,國立台灣師範大學化學系研究所,1999
    18. Frensdorff, H. K. Stability constants of cyclic polyether complexes with univalent cations. J. Am. Chem. Soc. 1971, 93, 600-606.
    19. Satsuo, K.; Ajay, B.; Yumi, F.; Hiroyuki, M. Copper(II)-selective electrode using thiuram disulfide neutral carriers. Anal. Chem. 1988, 60, 2464-2467.
    20. Cox, B. G.; Schneider, H.; Strroka, J. Kinetics of alkali metal complex formation with cryptands in methanol. J. Am. Chem. Soc. 1978, 100, 4746-4749.
    21. Richard, S. H.; Leonidas, G. B. Nitrate-selective electrode developed by electrochemically mediated imprinting/doping of polypyrrole Anal. Chem. 1995, 67, 1654-1660
    22. Saul Patai and Zvi Rappoport “Crown Ethers and Analogs”, 1989, John Wiley & Sons, U.S.A.
    23. Simmons, H. E.; Park, C. H. Macrobicyclic amines. I. Out-in isomerism of 1,(k+2)-diazabicyclo[k.l.m]alkanes J. Am. Chem. Soc. 1968, 90, 2428-2429.
    24. C. S. Chiou and J. S. Shih Multifunctional Cryptand Coated Piezoelectric Quartz Crystal Detector for Cations, Anions and Organic Molecules, Anal. Chim. Acta. 1999, 392, 125-133
    25. L. F. Wei and J. S. Shih Fullerene-Cryptand Coated Piezoelectric Crystal Urea Sensor Based on Urease, Anal. Chim. Acta. 2001, 437, 77-85
    26. Anthony, P. D.; John, F. G.; Justin, J. P. Angew. Chem. Int. Ed. Engl. 1996, 35, 1312-1315.
    27. C. S. Chiou; J. S. Shih, Bifunctional cryptand modifier for capillary electrophoresis in separation of inorganic/organic anions and inorganic cations. Analyst 1996, 121, 1107-1110.
    28. 呂鋒洲; 林仁混. 基礎酵素學. 1991.
    29. 陳國誠. 微生物酵素工程學. 1989
    30. W. Chuang and J. S. Shih, Preparation and Application of Immobilized-Glucose Oxidase Enzyme in Fullerene C60-Coated Piezoelectric Quartz Crystal Glucose Sensor, Sensors & Actuators 2001, 81(1), 1-8
    31. M. S. Chang and J. S. Shih, Fullerene-Cryptand Coated Piezoelectric Crystal Membrane Glucose Enzyme Sensor, Sensors & Actuators, 2000, 67, 275-281
    32. Chang, H. C.; Chen, P. Y.; Cheng, A. J. Enzyme immobilization and polymer Material. Chemistry. 1994, 52, 237-243.
    33. 陳治誠. 生化感測器技術簡介. 科儀新知. 1993, 15(2), 71-81.
    34. Bowers, Larry D. Application of immobilized biocatalysts in chemical analysis. Anal. Chem. 1986, 58(4), 513A, 514A, 516A, 518A, 520A, 522A, 524A, 526A, 528A, 530A.
    35. Liu, B.; Hu, R; Deng, J. Fabrication of an amperometric biosensor based on the immobilization of glucose oxidase in a modified molecular sieve matrix. Analyst. 1997, 122, 821-826
    36. Xinhao Yang, Lin Hua, Haiqing Gong, Swee Ngin Tan Covalent immobilization of an enzyme (glucose oxidase) onto a carbon sol–gel silicate composite surface as a biosensing platform. Analytica Chimica Acta 2003, 478, 67-75.
    37. Herdan, J. M.; Balulescu, M.; Cira, O. Enantioselective hydrolysis of racemic esters using pig liver esterase. Journal of Molecular Catalysis A:Chemical. 1996, 107, 409-414.
    38. Nursel Pekel, Bekir Salih, Olgun Güven Activity studies of glucose oxidase immobilized onto poly(N-vinylimidazole) and metal ion-chelated poly(N-vinylimidazole) hydrogels. Journal of Molecular Catalysis B: Enzymatic 2003, 21, 273-282
    39. Higuchi, A.; Hara, M.; Yun, K. S.; Tak, T. M. Recognition of substrates by membrane potential of immobilized glucose oxidase membranes. Journal of Applied Polymer Science. 1994, 51, 1735-1739.
    40. Godjevargova, Ts.; Dimov, A.; Vassileva, N. Effects of chemical modifications and immobilization of glucose oxidase onto acrylonitrile copolymer membranes on membrance potential and membrance charge density Journal of Membrane Science. 1996 , 116, 273-278.
    41. Sinan Akgol, Handan Yavuz, Serap S¸ enel, Adil Denizli Glucose oxidase and catalase adsorption onto Cibacron Blue F3GA-attached microporous polyamide hollow-fibres Reactive & Functional Polymers 2003, 55, 45-51
    42. Allen J. Bard& Larry R. Faulkner. “Electrochemical Method”, 1980, John Wiley& Sons, U.S.A.
    43. Aodhmer, C.; Zhiqiang, G.; Andrzej, L.: Ari, I. All-solid-state sodium-selective electrode based on a calixarene ionophore in a poly(vinyl chloride) membrane with a polypyrrole solid contact. Anal. Chem.1992, 64, 2496-2501
    44. Suresh, K. S.; Vinod, K. G.; Suresh, PVC-based 2,2,2-cryptand sensor for zinc ions J. Anal. Chem. 1996, 68, 1272-1275.
    45. Jordi, B.; Sylvia, D.; Leonidas, G. B. Selective electrodes for silver and anions based on polymeric membranes containing complexes of triisobutylphosphine sulfide with silver. Anal. Chem. 1991, 63, 1585-1589.
    46. Maria de los A., Arada Pèrez, Leonel Marín, Josefina Calvo Quintana, Mehrdad Yazdani-Pedram, Influence of different plasticizers on the response of chemical sensor based on polymeric membranes for nitrate ion determination. Sensors & Actuators B 2003, 89, 262-268
    47. Vasile, V. C.; Miklos, E.; James, S. R.; Timothy, A. J.; Michael, R. N.; Richard, P. B. Aliphatic polyurethane as a matrix for pH sensors: effects of native sites and added proton carrier on electrical and potentiometric properties. Talanta 1996, 43, 143-151.
    48. Jain. A. K.; Gupta, V. K.; Singh, L. P.; Khurana, U. Macrocycle based membrane sensor for the determination of cobalt(II) ions Analyst 1997, 122, 583-586.
    49. Lee, H. J.; Hong, U. S.; Lee, D. K.; Shin, J. H.; Nam, H.; Cha, G. S. Solvent-processible polymer membrane-based liquid junction- free reference electrode Anal. Chem. 1998, 70, 3377-3383.
    50. Keplinger, E. J.; Jachimowicz, A.; Kohl, F. Water flux across neutral carrier membranes Anal. Chem. 1998, 70, 4271-4279.
    51. Kimura, K.; Sunagawa, T,.; Yajima, S.; Miyake, S.; Yokoyama, Y. Neutral carrier-type ion sensors based on sol-gel-derived membranes incorporating a bis(crown ether) derivative by covalent bonding. Anal. Chem. 1998, 70, 4309-4313.
    52. Marin, H.; Peter, M, G,; Werner, E. M.; Simon, W. Membrane technology and dynamic response of ion-selective liquid-membrane electrodes. Anal. Chem. 1991, 63, 1380-1386.
    53. T. Y. Wang and J. S. Shih, Iron (III) Ion Selective Electrode Based on Dithia Crown Ethers, J. Chin. Chem. Soc. 1988, 35, 405
    54. D. Wang and J. S. Shih, Cesium Ion Selective Electrode Based on 15-Crown-5-PW, Analyst 1985, 110, 635
    55. M. T. Lai and J. S. Shih, Mercury (II) and Silver (I) Ion Selective Electrodes Based on Dithia Crown Ethers, Analyst, 1986, 111, 891
    56. S. R. Sheen and J. S. Shih, Lead (II) Ion Selective Electrodes Based on Crown Ethers, Analyst, 1992, 117, 1679
    57. Yuan, R.; Chai, Y. Q.; Liu, D.; Gao, D.; Li, J. Z.; Yu, R. Q. Schiff base complexes of cobalt(II) as neutral carriers for highly selective iodide electrodes Anal. Chem. 1993, 65, 2572-2575.
    58. Stacy, A. O.; Sylvia, D.; Leonidas, G. B. Nitrogen oxide gas sensor based on a nitrite-selective electrode. Anal. Chem. 1991, 63, 1278-1281
    59. Richard, P. B.; Erno, L. Pure and Appl. Chem. 1994, 66, 2527-2536.
    60. Oesch, U.; Brzozka, Z.; Xu, A.; Rusterholz, B.; Suter, G.; Pham, H. V.; Welti, D. H.; Ammann, D.; Pretsch, E.; Simon, W. Design of neutral hydrogen ion carriers for solvent polymeric membrane electrodes of selected pH range. Anal. Chem. 1986, 58, 2285-2289.
    61. Michael, H. A.; Angela, F.; Danil, N.; Walter, H. L. A thermochemical cycle for the heat of complexing of Na+ and K+ cations with cryptand[2,2,2] in water and methanol. J. C. S. Chem. Comm. 1977, 893-894.

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