臺灣博碩士論文加值系統

目前在檢測血液或尿液中嗎啡量常用的方法是利用管柱層析如氣象層析儀和高效能液相層析儀兩種方法。但是這樣的方法中往往需要事前的樣品處理需要相當多的時間與人力，免疫方法可以解決管柱耗時的問題。本篇所用的方法是利用抗嗎啡抗體與標幟上嗎啡的G6PDH(Glucose-6-phosphate dehydrogenase labeled with morphine, G6PDH-morphine)在光學與石英壓電免疫感測器上來進行偵測。在免疫感測器的部分，有很好的線性關係，而且偵測下限是113 ng/ml，這樣的偵測下限遠低於目前法律規定的300ng/ml，因此可以應用在實際的檢驗上，且光學免疫感測器的相對標準偏差值(R.S.D.) 3%左右，這是不錯的再線性的檢測方法。並且本篇也探討體內血清與尿液中主要的成分對於這個感測方法的影響。在石英壓電免疫感測器上，首先製備C60-G6PDH並且把C60-G6PDH固定在石英壓電晶體上作為感測膜來對於溶液中抗嗎啡抗體，當C60-G6PDH的吸附溶液中抗嗎啡抗體後會造成頻率的下降來作為偵測的標準，利用固定C60-G6PDH的石英壓電晶體來偵測溶液中的抗嗎啡抗體有相當不錯的再現性且對於抗嗎啡抗體有不錯的選擇性。

Current technology for determination of morphine in blood or urine is often to use the chromatographic methods such as GC and HPLC, but most of these methods are time consuming. To solve this problem, a morphine optical immuno-sensor and a quartz piezoelectric crystal sensor based on anti-morphine antibody and Glucose-6-phosphate dehydrogenase labeled with morphine (G6PDH-morphine) for opiate solutions were designed and prepared in this study. The optical morphine sensor exhibited good sensitivity with a detection limit of 113 ng/mL which was far below the allowed concentration (300 ng/mL) in law for morphine in biological solutions. The optical sensor also showed a high reproducibility with a relative standard deviation (RSD) of approximately 3 %. The interference of major biological species in simulated serum and urine solutions for the determination of morphine with the optical immuno-sensor was investigated and discussed. In the preparation of the quartz piezoelectric sensor, immobilized fullerene C60 - G6PDH-morphine was synthesized and applied as a coating material on quartz piezoelectric crystals for the immunoassay of anti-morphine antibody in aqueous solutions. The oscillating frequency of the G6PDH-morphine coated-piezoelectric crystal of the piezoelectric sensor decreased by adsorption of anti-morphine antibody in solutions. The piezoelectric crystal sensor showed nearly no interference from various biological species in simulated serum. The piezoelectric crystal sensor based on immobilized C60-G6PDH-morphine for anti-morphine antibody also exhibited good sensitivity and good reproducibility.

英文摘要………………………………………………………………...I
中文摘要………………………………………………………………...II
目錄……………………………………………………………………..III
圖目錄……………………………………………………………….….III
表目錄……………………………...…………………………………VIII
第一章 緒論……………………………………………………………1
1-1 嗎啡簡介…………………………………………………………...1
1-1-1 藥物濫用………………………………………………………1
1-1-2 毒品種類的區分………………………………………………2
1-1-3 鴉片、嗎啡、海洛因簡介……………………………………3
1-1-4 嗎啡的藥理作用………………………………………………5
1-1-5嗎啡常用的分析方法………………………………………….9
1-1-6 法定嗎啡的分析方法………………………………………...13
1-2 免疫分析…………………………………………………………..13
1-2-1放射免疫分析法………………………………………………..14
1-2-2 螢光免疫分析法……………………………………………….15
1-2-3 酵素免疫分析法……………………………………………….15
1-2-3-1酵素連結免疫吸附法ELISA（Enzyme-link immunosorbent assay…………...………………………………………………………..16
(一)三明治型(sandwich technique)…………………………………….16
(二)競爭型（competitive technique）………………………………….17
1-2-3-2酵素增幅免疫分析法EMIT(Enzyme Multiplied Immunoassay Technique)…………………………………………..……………….….19
1-3 免疫感測器(Immunosasensors)………………………………..….23
1-3-1 壓電免疫感測器(Piezoelectric immunosasensors)…………….24
1-3-2 光學免疫感測器(optical immunosasensors)…………………...25
1-4 碳六十化學………………………………………………………...27
1-4-1 碳六十的發現…………………………………………..…….27
1-4-2 碳六十的基本性質……………………………………..…….28
1-4-3 碳六十的化學反應…………………………………………...32
1-5 光敏電阻…………………………………………………………...33
1-6 壓電晶體…………………………………………………………...36
1-6-1 壓電晶體之壓電性…………………………………………….36
1-6-2 石英振盪器…………………………………………………….36
1-6-3 石英微量天平(Quartz crystal microbalance, QCM)…………..39
1-6-4 石英振盪器的線路…………………………………………….43
1-6-5 石英壓電晶體在分析化學領域上的應用…………………….45
1-7 訊號對雜訊的加強(signal-to-noise enhancement)………………..50
1-7-1整體平均………………………………………………………….50
1-7-2箱型平均………………………………………………………….51
第二章 實驗部分………………………………………………………52
2-1藥品與儀器…………………………………………………………52
2-1-1 藥品…………………………………………………………...52
2-1-2 使用儀器……………………………………………………...52
2-2石英晶體的處理……………………………………………………53
2-2-1石英電極的表面塗佈液(coating solution)……………………..53
2-2-2表面塗佈法……………………………………………………..54
2-3 實驗系統…………………………………………………………..54
2-3-1 光學偵測系…………………………………………………...54
2-3-1-1光學偵測原理…………………………………………….54
2-3-1-2 自製光學偵測系統簡述…………………………………55
2-3-2 QCM系統…………………………..…………………………59
2-4 實驗裝置與實驗步驟……………………………………………...61
2-4-1 以光學方式偵測抗體抗原…………………………………….61
2-4-2 碳六十與酵素-嗎啡間作用力的研究…………………………62
2-4-3抗嗎啡抗體與酵素-嗎啡之間結合的研究…………………….63
第三章 實驗結果與討論………………………………………………64
3-1光學偵測系統偵測嗎啡研究………………………..……………..64
3-1-1抗嗎啡抗體與酵素-嗎啡溶液間作用………………………....64
3-1-2嗎啡-酵素及嗎啡抗體用量對電壓變化的影響………………70
3-1-3嗎啡濃度對於電壓改變的影響……….…….…………...……74
3-1-4光源對測量結果影響與光學感測器的再現性……..………...77
3-1-5體內常見干擾物對光學感測器的影響………….……………79
3-1-6 pH值對於偵測結果的影響…………………………………81
3-2利用石英壓電晶體偵測抗體………………………………………82
3-2-1碳六十與酵素-嗎啡間的作用力…………………………………82
3-2-2嗎啡抗體與嗎啡-酵素-之間的作用力……………….………….84
3-2-3碳六十/PVC塗佈量對嗎啡-酵素的效應…………………..……88
3-2-4嗎啡-酵素濃度對於C60/PVC塗佈壓電晶片頻率變化改變的影響………………………………………………………………………..89
3-2-5抗嗎啡抗體濃度對頻率改變所造成的影響………………….…90
3-2-6石英壓電晶體感測器的再現性………………………………….91
3-2-7血液中干擾物對壓電感測器的影響…………………………….92
第四章 結論……………………………………………………………94
參考文獻…………………………………………………………………a
附錄………………………………………………………………………e

1. 王學仕. 實用麻醉藥理學 1983
2. 謝瑞智等著.警察百科全書. 正中書局. 2000
3. 林信男. 藥物濫用 橘井文化1994
4. Wesley G.Clark 原著. 李勇進等譯. 醫用藥理學 九州出版 1989
5. 林松洲 應用藥理學 生韻出版 1983
6. 藥物濫用宣導教育防治手冊 行政院衛生署編印
7. 防治嗎啡海洛因宣導教育手冊 行政院衛生署編印
8. M. Manfred R.; S., Stefan; Kraemer, Thomas, Determination of drugs of abuse in blood, J. Chromatogr. B 1998, 713, 91-109
9. G. Sakai, K. Ogata, T. Uda, N. Miura, N. Yamazoe, A surface plasmon resonance-based immunosensor for highly sensitive detection of morphine. Sensors and Actuators B 1998, 49, 5-12
10. K. Aoki, T. Yoshida. Y. Kuroiwa, Forensic immunochemistry, Forensic Science International 1996, 80, 163-173
11. P. Lillsunde, L. Michelson, T. Forsstrom, T. Korte, E. Schultz, K. Ariniemi, M. Portman, M. Sihvonen, T. Seppala, Comprehensive drug screening in blood for detecting abuse drugs or drugs potentially hazardous for traffic safety, Forensic Science International 1996, 77, 191-210
12. R. B. Taylor, A. S. Low, R. G. Reid, Determination of opiates in urine by capillary electrophoresis, J. Chromatogr. B 1996, 675, 213-223
13. 濫用藥物尿液檢驗機構認可基準 行政院衛生署管制藥品管理局
14. 進階版生物技術 教育部科技顧問室
15. 通識版生物技術 教育部科技顧問室
16. 田蔚城. 生物技術的發展與應用 1996
17. H., Marie-Claire; B., Damià, Strengths and limitations of immunoassays for effective and efficient use for pesticide analysis in water samples: A review Analytica Chimica Acta, 1998, 362, 3-34
18. N.P. Groome, A. Tsigou, M. Cranfield, P.G. Knight, D.M. Robertson, Enzyme immunoassays for inhibins, activins and follistatins. Molecular and Cellular Endocrinology, 180, 2001, 73—77
19. D. Monroe, Enzyme immunoassay, Anal. Chem. 1984, 56, 921A
20. E. Engvall, P. Perlmann, Immunochemistry 1971, 8, 871
21. E. Engvall, K. Josson, Perlmann, P. Biochim. Biophys. Acta 1971, 251, 427
22. K. Rubenstein, E. et al. Biochem. Biophys. Res. Commun. 1972, 47, 846
23. 呂鋒洲. 林仁混 基礎酵素學 1991
24. D. A. Skoog, F. J. Holler, T. A. Nieman, Principle of instrumental analysis 5th edition, Saunders college publishing 1997
25. Peter B. Luppa, Lori J. Sokoll, Daniel W. Chan, mmunosensors─principles and applications to clinical chemistry, Clinica Chimica Acta 2001, 314,1—26.
26. G. Electra, L. Christopher R, Immunosensors, Current Opinion in Biotechnology, 1996, 7, 66-71.
27. Tohyama, O.; Kohashi, M.; Sugihara, M.; Itoh, H. A fiber-optic pressure microsensor for biomedical applications Sensors and Actuators A: Physical 1998, 66, 150-154
28. Muller, Raluca; Moagar Poladian, V.; Pavelescu, I.; Manea, Elena; Cristea, Dana; Obreja, Paula Technological processes and modeling of opto-electro-mechanical microstructures Materials Science in Semiconductor Processing 2000, 3, 427-431
29. H. W. Kroto, Heath, J. R.; O’Brien, S. C.; Curl, R. F., Smalley, R. E. C60: Buckminsterfullerene. Nature. 1985, 318, 162-163
30. Kratschmer. W., Lamb. L. D., Fostiropoulos, K., Huffman, D. R. Solid, C60 : a new form of carbon. Nature (London). 1990,374, 354-358
31. Hawkin, 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.
32. Withers, J. C.; Loutfy, R. O., Lowe, T. P. Fullerene Commercial Vision. Fullerene Science And Technology. 1997, 5(1), 1-31
33. 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
34. Kroto H. W., Allaf, A. W.; Balm, S. P. C60: Buckminsterfullerene. Chem. Rev. 1991, 91, 1213-1235
35. Chen, W.; Xu, Z. Temperature Dependence of C60 solubility in different solvent. Fullerence Science and Technology. 1998, 6(4), 695-705
36. 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, 8634-8636
37. Taylor, R.; Walton, D. R. M. The chemistry of Fullerenes. Nature. 1993, 363, 685-693
38. 鐘國家, 謝勝治, 感測器原理與應用實習 全華科技圖書, 1999
39. http://www.cds-lighting.com.tw/
40. 許書務. 光感測器介面專題製作 電子技術出版社
41. 紀培錦. 新電子科技雜誌 1989, 17, 196
42. 湯進德. 微電子界面技術 全華科技圖書 1984
43. C. K. O’Sullivan, G. C. Guibault Commercoal Quartz crystal microbalances-theory and applications. Biosensors and Bioelectronics. 1999, 14, 663-670
44. Geddes, L. A.; Baker, L. E. Principle of applied biochemical Instrumentation (3rd Ed.) John Wiley & Sons. New York. 1989,163
45. L. L. Levenson, ”Cimento”, Suppl.2, Ser.1, 1967, 5, 321.
46. C. Lu, C. A. W. Czanderna, Applications of piezoelectric quartz crystal microbalance. Elsevier Science. New York, 1984
47. Sauerbrey, G. Z. Z. Phys. 155, 206.
48. J. Hlavay, G. Guilbualt, Applications of the piezoelectric crystal detector in analytical chemistry. Anal. Chem. 1977,49, 1890-1898
49. W. H. King, Piezoelectric sorption detector. Anal. Chem., 1964, 36, 1735-1739
50. Mandelis, Christofides, Physics, chemistry and technology of solid state gas sensor devices. John Wiley & Sons. New York, 1993
51. M. Thompson, A. L. Kipling, W. D. Duncan-Hewitt, Thickness-shear-mode Acoustic Wave Sensors in the Liquid Phase A Review. Analyst. 1991, 116, 881-890
52. D. Binkley, R. Dessy, J. Chem. Educ. 1979, 56, 148
53. R. L. Rowell Data Manipulation and handling J. Chem. Educ. 1978, 55, 148
54. 白中和 RS-232技術詳解與應用 全華科技圖書 1997
55. M. Waite著原著. 林健毓譯. Microsoft QuickBasic Bible中譯本 1993
56. Peter B. Luppa a,), Lori J. Sokoll b, Daniel W. Chan, mmunosensors─principles and applications to clinical chemistry, Clinica Chimica Acta 2001, 314,1—26.
57. P. C. Jurs,* G. A. Bakken, and H. E. McClelland ; Computational Methods for the Analysis of Chemical Sensor Array Data from Volatile Analytes, Chemical Reviews; 2000; 100(7); 2649-2678
58. Moon Hee Choi, Moon Hwan Kim, Ho Choll Cho, Min Sun Kim, Eun Ah Lee, Insook Rhee Paeng, Geun Sig Cha, Enzyme-linked competitive binding assays for digoxin, Bull. Korean Chem. Soc. 2001,22,417-420
59. 陳志誠. 生化感測器技術簡介. 科儀新知. 1993, 15, 71-81
60. http://kerouac.pharm.uky.edu/HomeTest/elisa/
61. http://www.cds-lighting.com.tw/
62. Data sheet of CdS photoconductive cell, HAMAMATSU
63. Ronghua Yang, Kemin Wang,* Liping Long, Dan Xiao, Xiaohai Yang, and Weihong Tan A Selective Optode Membrane for Histidine Based on Fluorescence Enhancement of Meso-Meso-Linked Porphyrin Dimer, Anal. Chem., 2002, 74, 1088-1096