本研究合成碳六十(C60)-固定化酵素:碳六十-過氧化氫分解酵素及碳六十-葡萄糖氧化酵素，並研製一生物感測器。將塗佈碳六十的石英壓電晶體感測器作為研究碳六十與酵素(過氧化氫分解酵素及葡萄糖氧化酵素)之間作用力的工具，結果顯示酵素能被碳六十吸收並呈現不可逆的化學吸附現象。
將碳六十與酵素反應生成不溶水的碳六十-過氧化氫分解酵素及碳六十-葡萄糖氧化酵素，並以IR鑑定產物，將產物譜圖與碳六十及酵素的譜圖比較後，能夠得知產物是由碳六十及對應的酵素鍵結而成。碳六十-過氧化氫分解酵素及碳六十-葡萄糖氧化酵素經氧電極測試後，發覺仍具活性。為方便重複使用，將碳六十-酵素塗佈於孔徑大小約5~10μm的多孔性二氧化矽材質上。本研究使用氧電極對這兩種固定化酵素的性質做探討。碳六十-過氧化氫分解酵素的最適pH及最適溫度皆與游離過氧化氫分解酵素相同，但固定化後對酸鹼的容忍度較好，而熱穩定性較差。碳六十-葡萄糖氧化酵素的最佳pH為7，有向鹼移動的趨勢。最適溫度為30度C，比游離的葡萄糖氧化酵素(35度C)低，另外，固定化後，酸鹼值及溫度的影響皆變小。碳六十-過氧化氫分解酵素使用95天後，仍保有93%的高活性。而碳六十-葡萄糖氧化酵素使用93天後，仍保有88%的高活性。
利用葡萄糖酸(葡萄糖經碳六十－葡萄糖氧化酵素催化後的產物)能被碳六十吸附的原理，使用自行合成的碳六十－葡萄糖氧化酵素研製一葡萄糖壓電感測器。隨著碳六十－葡萄糖氧化酵素量的增加，本感測器的靈敏度提高且具有較佳的偵測下限。本系統可應用於血糖的偵測上。

Immobilized fullerene(C60)-enzymes, e.g. C60-catalase and C60-glucose oxidase, were synthesized and applied in biological detection systems for hydrogen peroxide and glucose respectively. The C60-coated piezoelectric quartz crystal sensor was employed to study the interaction between C60 and enzymes. The chemisorption between C60 and some enzymes, e.g. catalase and glucose oxidase, was found.
Water insoluble C60-catalase and C60-glucose oxidase were identified with IR spectrography. These immobilized enzymes were still activated. The porous SiO2 material (pore size was about 5~10μm) was coated with C60-enzymes for reuses. The properties of both immobilized C60-catalase and C60-glucose oxidase were investigated with the O2 electrode. Optimum pH and optimum temperature of immobilized C60-catalase were the same with solvated catalase. Immobilized C60-catalase showed better stability toward pH variation but less thermal stability than solvated catalase. The life time of immobilized C60-catalase was long enough with an activity of 93% after 95 days. Optimum pH of immobilized C60-glucose oxidase at 7 and optimum temperature at 30oC were found while optimum pH was at 5.1 and optimum temperature was at 35oC for solvated glucose oxidase. Immobilized C60-glucose oxidase showed better stability toward pH variation and better thermal stability than solvated glucose oxidase. The life time of immobilized C60-glucose oxidase was also long enough with an activity of 88% after 93 days.
The C60 coated piezoelectric crystal sensor with immobilized C60-glucose oxidase was also prepared to detect glucose in solutions. The home-made glucose PZ sensor was based on the adsorption of gluconic acid (the production of glucose oxidation reaction with C60-glucose oxidase) onto the C60 coated crystal. The glucose PZ sensor can be used for detection of human blood sugar.

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. Krätschmer, W.; Lamb, L. D.; Fostiropoulos, K.; Huffman, D. R. Solid C60 : a new form of carbon. Nature(London). 1990, 347, 354-358.
3. 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.
4. Withers, J. C.; Loutfy, R. O.; Lowe, T. P. Fullerene Commercial Vision. Fullerence Science And Technology. 1997, 5(1), 1-31.
5. Scrivens, W. A.; Bedworth, P. V.; Tour, J. M. Purification of Gram Quantities of C60. A New Inexpensive and Facile Method. J. Am. Chem. Soc. 1992, 114, 7917-7919.
6. Kroto, H. W.; Allaf, A. W.; Balm, S. P. C60: Buckminsterfullerene. Chem. Rev. 1991, 91, 1213-1235.
7. Chen, W.; Xu, Z. Temperature Dependence of C60 Solubility in Different Solvent. Fullerence Science And Technology. 1998, 6(4), 695-705.
8. 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.
9. Taylor, R.; Walton, D. R. M. The chemistry of fullerenes. Nature. 1993, 363, 685-693.
10. Moriguchi, T.; Yano, K.; Hokari, S.; Sonoda, M. Effect of repeated application of C60 combined with UVA radiation onto hairless mouse back skin. Fullerene Sci. Technol. 1999, 7(2), 195-209.
11. Al-Mohamad, A.; Allaf, A. W. Fullerene-60 thin films for electronic applications. Synthetic Metals. 1999, 104, 39-44.
12. 呂鋒洲; 林仁混. 基礎酵素學. 1991.
13. 陳國誠. 微生物酵素工程學. 1989.
14. Chang, H. C.; Chen, P. Y.; Cheng, A. J. Enzyme Immobilization and Polymer Material. Chemistry. 1994, 52, 237-243.
15. 陳治誠. 生化感測器技術簡介. 科儀新知. 1993, 15(2), 71-81.
16. Bowers, L. D. Application of immobilized biocatalysts in chemical analysis. Anal. Chem. 1986, 58(4), 513A, 514A, 516A, 518A, 520A, 522A, 524A, 526A, 528A, 530A.
17. 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.
18. Spagna, G.; Pifferi, P. G.; Gilioli E. Immobilization of a pectinlyase from Aspergillus niger for application in food technology. Enzyme Microb. Technol. 1995, 17, 729-738.
19. Alkorta, I.; Garbisu, C.; Llama, M. J.; Serra, J. L. Industrial applications of pectic enzymes : a review. Process Biochemistry. 1998, 33(1), 21-28.
20. Xu, C. X.; Marzouk, S. A. M.; Cosofret, V. V.; Buck, R. P.; Neuman, M. R.; Sprinkle, R. H. Development of a diamine biosensor. Talanta. 1997, 44, 1625-1632.
21. Mazzei, F.; Botrè, F.; Botrè, C. Acid phosphatase/glucose oxidase-based biosensors for the determination of pesticides. Analytica Chimica Acta. 1996, 336, 67-75.
22. Reiss, M.; Heibges, A.; Metzger, J.; Hartmeier, W. Determination of BOD-values of starch-containing waste water by a BOD-biosensor. Biosensors & Bioelectronics. 1998, 13, 1083-1090.
23. Wittstock, G.; Schuhmann, W. Formation and Imaging of Microscopic Enzymatically Active Spots on an Alkanethiolate-Covered Gold Electrode by Scanning Electrochemical Microscopy. Anal. Chem. 1997, 69, 5059-5066.
24. Bulmuş, V.; Ayhan, H.; Pişkin, E. Modified PMMA monosize microbeads for glucose oxidase immobilization. The Chemical Engineering Journal. 1997, 65, 71-76.
25. Kang, E. T.; Neoh, K. G.; Huang., S. W.; Lim, S. L.; Tan, K. L. Surface-Functionalized Polyaniline Films. J. Phys. Chem. B. 1997, 101, 10744-10750.
26. Ukeda, H.; Fujita, Y.; Ohira, M.; Sawamura, M. Immobilized Enzyme-based Microtiter Plate Assay for Glucose in Foods. J. Agric. Food Chem. 1996, 44, 3858-3863.
27. Turmanova, S.; Trifonov, A.; Kalaijiev, O.; Kostov, G. Radiation grafting of acrylic acid onto polytetrafluoroethylene films for glucose oxidase immobilization and its application in membrane biosensor. Journal of membrane Science. 1997, 127, 1-7.
28. Alves Da Silva, M. Helena Gil, J. S. Redinha, Ana M. Oliveira Brett, J. L. Costa Pereira. Immobilization of glucose oxidase on Nylon Membranes and Its Application in a Flow-Through Glucose Reactor. Journal of Polymer Science :Part A: Polymer Chemistry. 1991, 29, 275-279.
29. Audebert, P.; Demaille, C. Electrochemical Probing of the Activity of Glucose Oxidase Embedded Sol-Gel Matrices. Chem. Mater. 1993, 5, 911-913.
30. Künzelmann, U.; Böttcher, H. Biosensor properties of glucose oxidase immobilized within SiO2 gels. Sensors and Actuators B. 1997, 38-39, 222-228.
31. 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.
32. 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.
33. 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.
34. Eremin, A. N.; Otyutskii, S. V.; Metelitsa, D. I. Properties of Catalase Immobilized on Cellulose in Aqueous and Micellar Media. Kinetics and Catalysis. 1995, 36(6), 776-784.
35. Arica, M. Y.; Denizli, A.; Salih, B.; Piskin, E.; Hasirci, V. Catalase adsorption onto Cibacron Blue F3GA and Fe(Ⅲ)-derivatized poly(hydroxyethyl methacrylate) membranes and application to a continuous system. Journal of Membrane Science. 1997, 129, 65-76.
36. Lu, C.; Czanderna, A. W. Applications of Piezoelectric Quartz Crystal Microbalance. Elsevier Science. New York. 1984.
37. 吳朗. 電子陶瓷-壓電. 全欣科技圖書. 1994.
38. 吳朗. 感測與轉換原理,元件與應用. 全欣科技圖書. 1992.
39. Ikeda, T. Fundamentals of piezoelectricity. Oxford. Sci. Publ. 1990.
40. Geddes, L. A.; Baker, L. E. Principle of Applied Biomedical Instrumentation.(3rd Ed.) John Wiley & Sons. New York. 1989, 163.
41. Martin, S. J.; Frye, G. C.; Ricco, A. J. Effect of Surface Roughness on the response of Thickness-Shear Mode Resonators in Liquids. Anal. Chem. 1993, 65, 2910-2922.
42. 紀培錦. 新電子科技雜誌. 1989, 17, 196.
43. 湯進德. 微電子界面技術. 全華科技圖書. 1984.
44. 袁帝文; 黃柏鈞. 數位邏輯設計與分析. 全欣科技圖書. 1992.
45. 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.
46. Hlavay, J.; Guilbault, G. G. Applications of the Piezoelectric Crystal Detector in Analytical Chemistry. Anal. Chem. 1977, 49(13), 1890-1898.
47. Sauerbrey, G. Z. Z. Phys. 1959, 155, 206.
48. William H. King, Jr. Piezoelectric Sorption Detector. Anal. Chem. 1964, 36(9), 1735-1739.
49. Mandelis and Christofides. Physics, Chemistry and Technology of Solid State Gas Sensor Devices. John Wiley & Sons. New York, 1993.
50. Ruys, D. P.; Andrade, J. F.; Guimarães, O. M. Mercury detection in air using a coated piezoelectric sensor. Analytica Chimica Acta. 2000, 404, 95-100.
51. Chang, P.; Shih, J. S. Multi-channel piezoelectric quartz crystal sensor for organic vapours. Analytica Chimica Acta. 2000, 403, 39-48.
52. Barkó, G.; Hlavay, J. Application of an artificial neural network (ANN) and piezoelectric chemical sensor array for identification of volatile organic compounds. Talanta. 1997, 44, 2237-2245.
53. Bruckenstein, S.; Shay, M. Experimental Aspects of Use of the Quartz Crystal Microbalance in Solution. Electrochimica Acta. 1985, 30(10), 1295-1300.
54. Thompson, M.; Kipling, A. L.; Duncan-Hewitt, W. C. Thickness-shear-mode Acoustic Wave Sensors in the Liquid Phase A Review. Analyst. 1991, 116, 881-890.
55. Chang, H. C.; Yang, C. C.; Yeh, T. M. Detection of lipopolysaccharide binding peptides by the use of a lipopolysaccharide-coated piezoelectric crystal biosensor. Analytica Chimica Acta. 1997, 340, 49-54.
56. Chu, X.; Jiang, J. H.; Shen, G. L.; Yu, R. Q. Simultaneous immunoassay using piezoelectric immunosensor array and robust method. Analytica Chimica Acta. 1996, 336, 185-193.
57. Caruso, F.; Rodda, E.; Furlong, D. N.; Niikura, K. Quartz Crystal Microbalance Study of DNA Immobilization and Hybridization for Nucleic Acid Sensor Development. Anal. Chem. 1997, 69(11), 2043-2049.
58. Gizeli, E.; Liley, M.; Lowe, C. R.; Vogel, H. Antibody Binding to a Functionalized Supported Lipid Layer: A Direct Acoustic Immunosensor. Anal. Chem. 1997 ,69(23), 4808-4813.
59. Abad, J. M.; Pariente, F.; Hernández, L.; Lorenzo, E. A quartz crystal microbalance assay for detection of antibodies against the recombinant African swine fever virus attachment protein p12 in swine serum. Analytica Chimica Acta. 1998, 368, 183-189.
60. Godjevargova, T.; Konsulov, V.; Dimov, A. Preparation of an ultrafiltration membrane from the copolymer of acrylonitrile-glycidylmethacrylate utilized for immobilization of glucose oxidase. Journal of Membrane Science. 1999, 152, 235-240.
61. Liu, B.; Hu, R.; Deng, J. Characterization of Immobilization of an Enzyme in a Modified Y Zeolite Matrix and Its Application to an Amperomertic Glucose Biosensor. Anal. Chem. 1997, 69, 2343-2348.