臺灣博碩士論文加值系統

有機磷劑是目前農業上最普遍使用的農藥之一，雖然它的半衰期短，環境殘留性也不高，但卻會造成人體內乙醯膽鹼酯酵素活性的阻礙而產生急毒性症狀。
本研究主要應用Sol-gel固化技術所發展的光纖型生物感測器來偵測有機磷農藥，其中以對有機磷農藥相當靈敏的乙醯膽鹼酯酵素作為生物活性物質，配合螢光酸鹼指示劑及光纖傳導元件，以達到快速準確分析有機磷農藥的目的。本研究中同時也進行螢光酸鹼指示劑種類、溶膠溶液中各添加液的配比、酵素-染劑溶液對溶膠溶液配比、基質濃度以及固化與基質用緩衝溶液之強度及pH值等參數進行最佳化的探討。
研究發現FITC-dextrans螢光染劑對pH值的靈敏度最高且流失率低，相當適用於連續式的系統。在最佳化方面，溶膠溶液中以TMOS：HCl：H2O = 1：3.6’10-5：2的配比最為適當，而酵素-染劑溶液對溶膠溶液的配比則在考慮固化時間與sol-gel脆裂的問題後選擇3~5之間，固化用緩衝溶液以5mM pH8.9 Tris-HCl能保持較高的初始螢光強度，基質用緩衝溶液則選定為1mM pH8.5 Tris-HCl，且基質濃度必須在50mM以下才不會造成基質溶液pH值的大幅改變。另外，實驗結果亦顯示，自行設計的連續式系統具有相當好的穩定性，經8次重複實驗其相對標準偏差值(RSD)為3.5%，而且對於基質(ACh)亦具有相當高的靈敏度，線性範圍介於0.5mM ~ 20mM之間(R2 = 0.98)。在有機磷農藥的添加測試部分，發現添加152ppb的paraoxon可得到30%的明顯抑制現象，說明自行設計的有機磷光纖生物感測器的確可用於有機磷農藥的即時分析。

Organophosphorus pesticides (OPPs) are widely used as insecticides for agricultural or household purposes since 1970s. Although they can be quickly decomposed in the environment, their highly acute toxicity to acetylcholinesterase (AChE) received highly attention. Therefore, the development of high efficient analytical techniques for real-time monitoring the residues of OPPs in the environments is needed.
The purpose of this study is to apply the sol-gel immobilization technology for developing a fiber-optic biosensor using AChE as biological recognition molecule for the detection of OPPs. Eight kinds of fluorescence dyes were used as the candidates for pH sensitive indicators. Also, the optimization in sol-gel formation and substrate preparation will be included.
Results obtained in this study show that FITC-dextrans is a suitable pH sensitive fluorescence indicator for AChE based biosensor due to its low leaching ability in continuos system. The optimized ratio of TMOS:HCl:H2O was 1:3.6’10-5:2. The mixing ratio of enzyme-dye solution and sol solution was in the range of 3 to 5. By using 5mM pH8.9 Tris-HCl as immobilization buffer, a higher initial fluorescent intensity was observed. The buffer capacity for substrate preparation was 1mM pH8.5 Tris-HCl and the substrate concentration must below 50mM to get a stable pH value. Moreover, this homemade fiber-optic biosensor was highly stable in analyzing the acetylcholine with the relative standard deviation (RSD) of 3.5% (n=8). A good linearity of acetylcholine in the range from 0.5 to 20mM was also obtained (R2=0.98). A 30% inhibition can be achieved when 152ppb paraoxon was added into the system. This suggests the developed biosensor system is suitable for real-time analysis of organophosphorus pesticides.

中文摘要…..……………………………………………………………I
英文摘要………………………………………………………………II
目錄…………………..…………………………………………….…III
圖目錄……………………………………………………………….VII
表目錄…………………………………………………………………XI
第一章 緒論………………………………………………………..…1
1-1 研究背景…………………………………………………..…1
1-2 研究目的……………………………………………………..4
第二章 背景與理論依據………………………………………….…6
2-1 有機磷農藥簡介……………………………………..………6
2-1-1 有機磷農藥之基本特性………………….…….…….6
2-1-2 有機磷農藥之生物毒性………………….……..….…9
2-1-3 有機磷農藥之環境限值………………………..…….13
2-2 生物感測器簡介………………………………..………..…17
2-2-1 生物感測器之基本架構……………………..………17
2-2-2 生物感測器之市場潛力………………………..……21
2-2-3 生物感測器之未來發展……………………..………24
2-3 有機磷生物感測器………………………….……….……..29
2-3-1 有機磷生物感測器簡介……….…………………….29
2-3-2 有機磷電化學生物感測器…………………………..30
2-3-3 有機磷光學生物感測器………………………..……33
2-3-4 目前有機磷生物感測器之種類及偵測極限………..37
2-4 Sol-gel固化技術………………………………………….…43
2-4-1 Sol-gel固化技術之簡介………………………….…..43
2-4-2 Sol-gel固化技術之基本原理………………………..44
2-4-3 本研究採用之sol-gel固化方法…………………….47
2-5 反應酵素動力機制…………………………………………48
第三章 研究方法與設備…………………….……………………..53
3-1 研究規劃………………………………..………………..…53
3-2 實驗設備………………………………..………..…………55
3-3 實驗藥品……………………………..……...……………...56
3-4 實驗系統架設………………………………………………57
3-5 實驗步驟……………………………………………………60
3-5-1 實驗溶液配製……………………………………….60
3-5-2 Sol-gel固化步驟……………………….………….…61
3-6 螢光指示劑的光學系統……………………………………61
3-6-1 Acridine的光學系統…………………………………61
3-6-2 FITC的光學系統…………………………………….62
第四章 結果與討論……………………………………………..….64
4-1 螢光酸鹼指示劑的選擇………………………………….…64
4-1-1 螢光染劑的基本特性測試…………….…….………64
4-1-2 pH值對螢光光譜的影響…………………….………65
4-2 Lab View程式的設計……………………………………….71
4-3 Acridine與FITC適用性的探討……………….…………..74
4-3-1 Acridine與FITC的流失率測試…………………….74
4-3-2 Acridine批次系統測試………………….……………76
4-3-3 Acridine對乙醯膽鹼酯酵素的毒性測試……………80
4-3-4 FITC對乙醯膽鹼酯酵素的毒性測試……………….80
4-4 Sol-gel固化技術的發展與最佳化………………………….84
4-4-1 Sol solution中TMOS、HCl與H2O之間的配比.….84
4-4-2 固化用緩衝溶液之pH值以及強度……………..….86
4-4-3 Sol solution與Enzyme-dye solution的混合比例……88
4-5 基質濃度與其緩衝溶液的最佳化…………………………89
4-5-1 基質用緩衝溶液之重要性………………….….……89
4-5-2 基質濃度的最佳化…………………………….…….92
4-5-3 基質溶液配製技術的發展………………….……….96
4-6 系統最佳化結果的總整理……………………….…………98
4-7 連續式系統測試………………………………..…………100
4-7-1 Sol-gel固化於反應器底部………………….………100
4-7-2 Sol-gel固化於套管頂端………………..…………..104
4-7-3 穩定性測試………………………….…………...…107
4-7-4 乙醯膽鹼檢量線的建立……………………….……109
4-7-5 酵素動力機制探討…………………………………112
4-7-6 有機磷農藥添加測試………………………………115
第五章 結論與建議………………………………………………..117
參考文獻………………………………………………………….…119
附錄…………………………………………………………………119

吳先琪，八種劇毒農藥整體性風險評估，行政院農委會，民國八十二年。
張怡南，生物感測器，生物技術的發展與應用，九州圖書文物有限公司出版，田蔚城主編，民國八十六年。
黃定國，鄭武順，許瑞祺，簡介生物感測器及其未來方向，生物產業Bioindustry，1996, 7/4, 291.
陳建源，生物感測器之發展與應用，生物產業Bioindustry，1996, 4-3/4, 205.
廖龍盛，實用農藥(修訂七版)，民國七十一年。
Aizawa, M., Seyama, T., Fueki, K., Shiokawa, J., Suzuki, S., Proceedings of the International meeting of chemical Sensors, Fukuoka, Elsevier, Amsterdam, 1983, p.683
Altstein, M., Segev, G., Aharonson, N., Ben-Aziz, O., Turniansky A., Avnir, D., Sol-gel entrapped cholinesterase: A microtiter plate methods for monitoring anti-cholinesterase compounds, J. Agric. Food. Chem., 1998, 46, 3318-3324.
Alvarez-Icaza, M., Bilitewski, U., Mass production of biosensor, Anal. Chem., 1993, 65/11, 525A-533A.
Andres. R.T., Narayanaswamy, R., Talanta, 1997, 44, 1335-1352.
Ariga, O., Suzuki, T., Sano, Y., Murakami, Y., Immobilization of a thermostable enzyme using a sol-gel preparation method, Journal of Fermentation and Bioengineering, 1996, 82/4, 341-345.
Ayyagari, M.S., Kamtekar, S., Pande, R., Marx, K.A., Kumar, J., Tripathy, S.K., Akkara, J., Kaplan, D.L., Chemiluminescence-based inhibition kinetics of alkaline phosphatase in the development of a pesticide biosensor, Biotechnol. Prog., 1995, 11, 699-703.
Bernabei, M., Chiavarini, S., Cremisini, C., Mascini M.,Palleschi, G., Analitical Letters, 1991, 24, 1317.
Bernabei, M., Chiavarini, S., Cremisini C., Palleschi, G., Biosens. Bioelectron., 1993, 8, 265.
Blum, L.J., Coulet, P.R., Biosensors principles and applications, Marcel Dekker Inc., 1991
Braun, S., Rappoport, S., Zusman, R., Avnir D., Ottolenghi, M., Biochemically active sol-gel glasses: the trapping of enzymes, Materials Letters, 1990, 10/1,2, 1-5.
Campanella, L., Achilli, M., Sammartino, M.P., Tomassetti, M., Bioelectrochemistry and Bioenergentics, 1991, 26, 237-249.
Campanella, L., Cocco, R., Sammartino M.D., Tomassetti, M., Sci. Total. Environ., 1992, 123/124, 1
Clark, L.C., Lyons, C., Electrode system for continuous monitoring in cardiovascular surgery, Ann. N.Y. Acad. Sci., 1962, 102, 29-33.
Chen, Z., Kaplan, D.L., Yang, K., Kumar, J., Marx K.A., Tripathy, S.K., Phycobiliproteins encapsulated in sol-gel glass, Journal of Sol-gel Science and Technology, 1996, 7, 99-108.
Cremlyn, R., Pesticides-preparation and mode of action. Wiley, Chicester, 1978, p.213.
Danzer, T., Schwedt, G., Chemotric methods for development of a biosensor system and the evaluation of inhibition studies with solutions and mixtures of pesticides and heavy metals. Part 1. Development of an enzyme electrodes system for pesticide and heavy metals screening using selected chemometric methods, Analytica Chimica Acta, 1996, 318, 275-286.
Dave, B.C., Dunn, B., Valentine J.S., Zink, J.I., Sol-gel ecapsulation methods for biosensors, Analytical Chemistry, 1994, 66/22, 1120A-1127A.
Dosoretz, C., Armon, R., Starosvetzky J., Rothschild, N., Entrapment of parathion hydrolase from Pseudomonas spp. In sol-gel glass, Journal of Sol-gel Science and Technology, 1996, 7, 7-11.
Dumschat, C., Muller, H., Stein, K., Schwedt, G., Pesticide-sensitive ISFET based on enzyme inhibition, Analytica Chimica Acta, 1991, 252, 7-9.
Durand, P., Malevialle, J., Nicaud, J.M., J. Anal. Toxicol., 1984, 8, 112.
Ellerby, L.M., Nishida, C.R., Nishida, F., Yamanaka, S.A., Dunn, B., Valentine J.S., Zink, J.I., Encapsulation of proteins in transparent porous silicate glasses prepared by the sol-gel method, Science, 1992, 255/28, 1113-1115.
Eto, M., Organophosphorous pesticides: Organic and biological chemistry, CRC, Boca Raton, FL., 1974, p.123.
Everett W.R., Rechnitz, G.A., Mediated bioelectrocatalytic determination of organophosphorus pesticides with a tyrosinase-based oxygen biosensor, Anal. Chem, 1998, 70, 807-810.
Giang P.A., Hall, S.A., Enzymatic deterimation of organic phosphorus insecticides, Anal. Chem., 1951, 23/12, 1830-1834.
Goodson, L.H., Jocobs, W.B., Meth. Enzymol., 1976, 44, 647.
Gruss, R., Scheller, F., Shao, C.J., Liu, C.C., Analytical Letters, 1989, 22 , 1159.
Guilbault, G.G., Practical fluorescence, Theory, Methods, and Techniques, Marcel Dekker Inc., New York and Basel, 1973, Chapter 14, p599.
Guilbault, G.G., Nabi Rahni, M., In Analytical uses of immobilized biological compounds for detection, medical and industrial uses, (G. G. Guilbault, M. Mascini, Eds.), NATO ASI Ser. C Vol. 226, D. Reidel Publishing, Dordrecht, 1989, p.187.
Guo Y., Guadalupe, A.R., Screen-printable surfactant-iinduced sol-gel graphite composites for electrochemical sensors, Sensors and Actuators B, 1998, 46, 213-219.
Hench L.L., Ulrich, D.R., Ultrastructure processing of ceramics, glasses, and composites, John Wiley & Sons, Inc., 1984, Chapter 8, p.183.
Janata, J., Huber, R.J., Cohen, R., Kolesar, E.S., Aviat. Space Environ. Med., 1981, 52, 666.
Lan, E.H., Dunn, B., Valentine J.S., Zink, J.I., Encapsulation of the ferritin protein in sol-gel derived silica glasses, Journal of Sol-gel Science and Technology, 1996, 7, 109-116.
Li, J., Chia, L.S., Goh N.K., Tan, S.N., Silica sol-gel immolized amperometic biosensor for the determination of phenolic compounds, Analytica Chimica Acta, 1998, 362, 203-211.
Louis, J., Sode, K., Karube, I., Amperometric determination of choline and acetylcholine with enzymes immobilized in a photocross-linkable polymer, Analitica Chimica. Acta, 1989, 228, 49-53.
Martorell, D., Cespedes, F., Martinez-Fabregas E., Alegret, S., Analytica Chimica Acta, 1994, 290, 343.
Marty, J.L., Sode K.,Karube, I., Electroanalysis, 1992, 4, 249.
Marty, J.L., Mionetto, N., Noguer, T., Ortega F., Roux, C., Biosens. Bioelectron., 1993, 8, 273.
Mionetto, N., Marty J.L., Karube, I., Biosens. Biolectron., 1994, 9, 463.
Mulchandani, A., Muchandani, P., Kaneva, I., Chen, W., Biosensor for direct determination of organophosphate nerve agents using recombinant Escherichia coli with surface-expressed organophosphorus hydrolase. 1. Potentiometric microbial electrode, Anal. Chem., 1998, 70, 4140-4145.
Navas Diaz, A., Ramos Peinado, M.C., Sol-gel cholinesterase biosensor for organophosphourus pesticide fluorimetric analysis, Sensors and Actuators B, 1997, 38/39, 426-431.
Navas Diaz, A., Ramos Peinado M.C., Torijas Minguez, M.C., Sol-gel horseradish peroxidase biosensor for hydrogen peroxide detection by chemiluminescence, Analytica Chimica Acta, 1998, 363, 221-227.
Negh-Ngwainbi, J., Foley, P.H., Kuan, S.S., Guilbault, G.G., Parathion antibodies on piezoelectric crystals, J. Am. Chem. Soc., 1986, 108, 5444.
Newman, A., Ranking pesticide by environment impact, Environ. Sci. Technol., 1995, 29, 324A-326A.
Nyamsi Hendji, A.M., Jaffrezic-Renault, N., Martelet, C., Clechet, P., Shul'ga, A.A., Strikha, V.I., Netchiporuk, L.I., Soldatkin, A.P. Wlodarski, W.B., Analitica Chimica Acta, 1993, 281, 3-11.
Palleschi, G., Bernabei, M., Cremisini, C., Mascini, M., Determination of organophosphorus insecticides with a choline electrochemical biosensor, Sensors and Actuator B, 1992, 7, 513-517.
Rainina, E.I., Efremenco E.N., Varfolomeyev, S.D., The development of a new biosensor based on recombinant E. coli for the direct detection of organophosphorus neurotoxins, Biosensors and Bioelectronics, 1996, 11/10, 991-1000.
Ramanathan, K., Kamalasanan, M.N., Malhotra, B.D., Pradhan, D.R., Immobilization and characterization of lactate dehydrogenase on TEOS derived sol-gel films, Journal of Sol-gel Science and Technology, 1997, 10, 309-316.
Rogers, K.R., Valdes, J.J., Eldefrawi, M.E., Acetylcholine receptor fiber-optic evanescent fluorosensor, Analytical Chemistry, 1989, 182, 353-359.
Rogers, K.R., Cao, C.J., Valdes, J.J., Eldefrawi, A.T., Eldefrawi, M.E., Acetylcholinesterase fiber-optic biosensor for detection of anticholinesterase, Fundamental and Applied Toxicology, 1991, 16, 810-820.
Rogers, K.R., Gerlach, C.L., Enviromental biosensor, a status report, Environ. Sci. Technol., 1996, 30/11, 486A-491A.
Sethi, R.S., Transducer aspects of biosensors, Biosensors and Bioelectronics, 1994, 9, 243-264.
Skladal, P., Detection of organophosphate and carbamate pesticides using disposable biosensors based on chemically modified electrodes and immobilized cholinesterase, Analytica Chimica Acta, 1992, 269, 281-287.
Skladal, P., Pavlik, M., Fiala, M., Pesticide biosensor based on coimmobilized acetylcholinsterase and butyrylcholinesterase, Analytical Letters, 1994, 27/1, 29-40 .
Skladal, P., Fiala, P., Krejci, J., Detection of pesticides in the environment using biosensors based on cholinesterase, Intern. J. Environ. Anal. Chem., 1996, 65, 139-148.
Stoycheva, M., Analitical Letters, 1994, 27, 3065.
Tran-Minh C., Beaux, J., Anal. Chem., 1979, 51, 91-95.
Trettnak, W., Reininger, F., Zinterl E., Wolfbeis, O.S., Fiber-optic remote detection of pesticides and related inhibitors of the enzyme acetylcholinesterase, Sensors and Actuators B, 1993, 11, 87-93.
Turniansky, A., Avnir, D., Bronshtein, A., Aharonson N., Altstein, M., Sol-gel entrapment of monoclonal anti-atrazine antibodies, Journal of Sol-gel Science and Technology, 1996, 7, 135-143.
Updike, S.J., Hicks, G.P., The enzyme electrode, Nature 1967, 214, 986-988.
Wang, J., Prasad V.A. Pamidi and Park, D.S., Screen-printable sol-gel enzyme-containing carbon inks, Analytical Chemistry, 1996, 68, 2705-2708.
Wollenberger, U., Setz, K., Scheller, F.W., Loffler, U., Gopel W., Gruss, R., Sens. Actuators B, 1991, 4, 257.
Yang, S., Lu, Y., Atanossov, P., Wilkins E., Long, X., Talanta, 1998, 47, 735-743.