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研究生:陳春吉
研究生(外文):Chun-Ji Chen
論文名稱:自主性單層薄膜電極之阻抗分析與其在內毒檢測上之應用
論文名稱(外文):Impedance Analysis of SAM Modified Electrode and Its Application on Endotoxin Detection
指導教授:張憲彰
指導教授(外文):Hsien-Chang Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:醫學工程研究所
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:94
中文關鍵詞:脂多醣體抗生素交流電阻抗分析法LAL檢測法石英晶體微天秤
外文關鍵詞:LipopolysacchrideAntibioticAC impedance spectroscopyLAL assayQCM
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  • 被引用被引用:16
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革蘭氏陰性菌細胞壁外膜之脂多醣體 (lipopolysaccharide, LPS)又稱為內毒素 (endotoxin),在細菌死亡解體時會釋放出來,可引起人體產生休克或內毒素血症,嚴重者甚至可導致死亡。目前LPS檢測以鱟試驗法(Limulus amebocyte lysate, LAL)最常被使用,其檢測極限可約達到2 ng/ml。但此法仍有靈敏度低和檢測費用昂貴,以及原料來源取得困難等問題,本研究擬以電極表面的修飾技術配合交流電阻抗分析法(ACIS),發展高靈敏度、成本低廉以及檢測程序簡便之LPS檢測系統。
實驗中我們以DTBA及PmB為感測元件進行LPS之檢測,亦即以自主性吸附 (SAM)之修飾程序,先將金電極浸泡於4,4’-dithiodi-(n-butyric acid) (DTBA)溶液中,使金電極表面產生帶有COOH之官能基,而成為我們探討之第一種電極(DTBA/Au)。其原理乃藉DTBA與LPS間之親疏水性作用,使LPS能夠吸附在電極表面上,當LPS添加後所產生之阻抗曲線即以架設於常數相位元件(CPE)元件下之電路模型,模擬分析LPS的檢測結果,其可使電阻(Rt)與電容(CSAM)分別產生48.1 kΩ/ng/ml及32.4 nF/ng/ml之變化量與0~0.6 ng/ml的線性範圍。DTBA/Au電極若再經由1-ethyl-3-(3-dimethylamionpropyl) carbodiimide (EDC)與N-hydrosuccinimide (NHS)的活化後,將抗生素Polymyxin B (PmB)固定於金電極表面,即成為本研究探討之第二種PmB-DTBA/Au電極。此則透過抗生素PmB與LPS之間的正負電吸引力,進而達到檢測LPS之目的,且其Rt與CSAM也分別有81.6 kΩ/ng/ml及56.6 nF/ng/ml之高偵測靈敏度及0~0.6 ng/ml的線性範圍。以PmB-DTBA/Au電極經pH 2.5之glycine buffer酸洗後可達到重複使用之目的,然靈敏度會逐漸呈略微降低。
以PmB/DTBA/Au修飾之電極在飲用水及生理食鹽水中進行LPS之檢測,在含有1 ng/ml之LPS溶液中,可分別使CSAM產生66 nF/ng/ml及33.2 nF/ng/ml之電容效應。前者之電容效應為後者之二倍,可歸因於溶液中離子較低的飲用水,有較大之電容效應;反之在生理食鹽水中,因其有許多正負離子存在,就會有較小之電容值。食鹽水受限於電容值不明顯,難以有較大之電容值變化。故本研究處在離子成分較低之溶液中,會有較明顯之LPS檢測結果。最後我們也以石英晶體微天秤(quartz crystal microbalance, QCM)方式佐證LPS的檢測結果,以DTBA-PmB/Au修飾之晶片在10 ng/ml LPS的濃度下會有15 Hz的變化量。

Lipopolysaccharide (LPS) was also called endotoxin which existed in the outer membrane of cell wall of Gram negative bacteria, it could induce some diseases such as shock, endotoxin, or death. Limulus amebocyte lysate (LAL) assay was the most common method for detecting LPS, which has the detection limit of 2 ng/ml. However, this method still defeats in its low sensitivity, high cost and the rare resource. In this study, modified electrodes combined with A.C. impedance spectroscopy (ACIS) were designed to develop a simpler, economical, and sensitive detection system.
DTBA and Polymyxin B (PmB) were separately used as the sensing elements for detecting LPS. The DTBA/Au electrode was fabricated by immobilization of 4,4’-dithiodi(n-butyric acid) (DTBA) on the gold surface through self-assembled membrane (SAM) process, which was the first detective electrode in our study (DTBA/Au). As LPS was added, it caused to an interaction between DTBA and LPS. This electrical change was detected by impedance analysis with circuit model, which was combined with a constant phase element (CPE). As a result, the sensitivity of Rt and CSAM for detecting LPS could be obtained to be 48.1 kΩ/ng/ml and 32.4 nF/ng/ml, and the linear range was 0 to 0.6 ng/ml. Then, the DTBA/Au electrode was successively activated with 1-ethyl-3-(3-dimethylamionpropyl) carbodiimide (EDC) and N-hydrosuccinimide (NHS) and coupled with PmB, this electrode was to be our second electrode (PmB-DTBA/Au). The sensitivity of Rt and CSAM for detecting LPS was 81.6 kΩ/ng/ml and 56.6 nF/ng/ml, and the linear range was 0 to 0.8 ng/ml. The electrode could be reused by washing with glycine buffer (pH 2.5), however its sensitivity gradually showed to be not sensitive enough.
The system with water and normal saline was evaluated, and the sensitivity of CSAM for detecting LPS with PmB-DTBA/Au electrode was 66 nF/ng/ml and 33.2 nF/ng/ml, respectively. It could be attributed that normal saline solution with higher ions content induced lower variation of capacitance. Namely, the detection system in this study is high sensitive to LPS in solution with lower ions content. We evidenced the effect of detecting LPS by quartz crystal microbalance (QCM), and the sensitivity of DTBA-PmB/Au modified electrode was 15 Hz in 10 ng/ml LPS.

第一章緒論
1-1簡介
1-1-1脂多醣體的結構
1-1-2脂多醣體的致病機制
1-1-3脂多醣體的檢測
1-2生醫感測器
1-2-1生醫感測器的定義與發展
1-2-2生醫感測器的組成
1-3生物感測元件的修飾方法
1-3-1基盤先處理
1-3-2生物感測元件固定的方式
1-4交流電阻抗分析法
1-4-1阻抗量測技術之演進
1-4-2阻抗頻譜圖與電路模型的關連性
1-4-3交流阻抗分析法在檢測上的應用
1-5研究動機與目的
1-6實驗架構
第二章實驗原理與方法
2-1電化學檢測之原理
2-1-1交流阻抗分析法之檢測原理
2-1-2循環伏安法的原理
2-1-3石英晶體微天秤法法之檢測原理
2-2實驗設備材及試劑
2-2-1實驗設備
2-2-2實驗試劑
2-3實驗方法
2-3-1電極表面之修飾程序
2-3-2交流阻抗分析法之檢測流程
2-3-3石英晶體微天秤法之檢測流程
第三章結果與討論
3-1等效電路設計之確立
3-1-1由阻抗曲線反推適當之電路模型
3-1-2以電路模型示意電極表面之各種物理現象
3-2電解研磨對LPS檢測之影響
3-2-1以阻抗曲線的變化評估電解研磨對LPS檢測的結果
3-2-2由電路模型中各元件值評估電解研磨對LPS檢測的結果
3-2-3電解研磨之前處理條件探討
3-2-4評估各電解研磨條件清潔表面之效能
3-3DTBA修飾時間之探討
3-3-1DTBA以不同時間修飾金電極表面的飽和度探討
3-3-2以DTBA為感測元件進行LPS檢測
3-4EDC活化電極表面之COOH對LPS檢測之影響
3-5以EDC及NHS活化電極表面之COOH對LPS檢測之影響
3-6PmB-DTBA/Au電極之再現性評估
3-7PmB-DTBA/Au電極之再生使用
3-8PmB-DTBA/Au在模擬樣本之LPS檢測
3-9以石英晶體微天秤法進行LPS檢測的評估
第四章結論與展望
4-1阻抗分析在LPS檢測上之可行性探討
4-2未來發展與應用
參考文獻

[1] 李國鏞, 游若萩, 微生物學, 華香園出版, 49-144, 1996.
[2] 王貴譽, 大學微生物學, 曉園出版社, 13-31, 1993.
[3] J. Cohen, “The detection and interpretation of endotoxaemia”, Intensive Care Medicine, 26, 51-56, 2000.
[4] D. Petsch, F. B. Anspach,, “Review article- Endotoxin removal from protein solutions”, Journal of Biotechnology, 76, 97-119, 2000.
[5] M. G. Netea, M. V. Deuren, B. J. Kullberg, J. M. Cavaillon, “Does the shape of lipid A determine the interaction of LPS with Toll-like receptor?”, Trends in Immunology:TREIMM., 23, 135-139, 2002.
[6] D. Petsch, F. B. Anspach, “Endotoxin removal from protein solutions”, Journal of Biotechnology, 76, 97-119, 2000.
[7] A. P. Moran, M. M. Prendergast, B. J. Appelmelk, “Molecular mimicry of host structures by bacterial lipopolysaccharides and its contribution to disease”, FEMS Immunology and Medical Microbiology, 16, 105-115, 1996.
[8] O. Holst, A. J. Ulmer, H. Brade, H. D. Flad, E. T. Rietschel, “Biochemistry and cell biology of bacterial endotoxins”, FEMS Immunology and Medical Microbiology, 16, 83-104, 1996.
[9] R. Stephens, M. Mythen, “Endotoxin immunization”, Intensive Care Medicine, 26, 129-136, 2000.
[10] E. T. Rietschel, “Handbook of endotoxin”, Elsevier Science, 46-56, 1984.
[11] H. R. Buller, S. J. Deventer, A. Sturk, J. Levin, J. W. Cate, “Bacterial endotoxins pathophysiological effects, clinical significance, and pharmacological control”, Alarn R. Liss, 17-45, 1988.
[ 2] S. Srimal, N. Surolia, S. Balasubramanian, A. Surolia, “Titration calorimetric studies to elucidate the specificity of the interactions of polymyxin B with lipopolycaccharides and lipid A”, The Journal of Biochemistry, 315, 679-686, 1996.
[13] J. C. Haas, P. J. Haas, P. M. Kessel, A. G. Strijp, “Affinities of different proteins and peptides for lipopolysaccharide as determined by biosensor technology”, Biochemical and Biophysical Research Communications, 252, 492-496, 1998.
[ 4] S. A. David, S. K. Awasthi, P. Balaram, “The role of polar and facial amphipathic character in determining lipopolysaccharide-binding properties in synthetic cationic peptides”, Journal of Endotoxin Research, 6, 249-256, 2000.
[ 5] R. S. Conrad, C. Galanos, “A new technique to analyze for Polymyxin B and its nonapeptide derivative”, Proceedings of the Oklahoma Academy of Science, 75, 51-56, 1995.
[16] 丁慶雄編譯, 免疫學第三版, 藝軒圖書出版社, 50-58, 1994.
[ 7] 王依蕾編譯, 實用免疫學, 藝軒圖書出版社, 47-59, 1994.
[ 8] T. J. Novitsky, P. F. Roslansky, G. R. Siber., “Turbidometric method of quantifying serum inhibition of Limulus amoebocyte lysate”, The Journal of Infectious Diseases, 20, 211-216, 1985.
[ 9] G. K. Lindsay, P. .F. Roslansky, “Single-step, chromogenic Limulus amoebocyte lysate assay for endotoxin”, Journal of Clinical Microbiology, 27, 947-951, 1989.
[20] K. Takzhzshi, M. Fukzdz, M. Kzzi, T. Yokochi, “Detection of lipopolysaccharide(LPS) and identification of its serotype by an enzyme-linked immunosorbent assay (ELISA) using poly-L-Lysine”, Journal of Immunology Method, 150, 67-71, 1992.
[21] E. A. James, K. Schmeltzer, F. S. Ligler, “Detection of endotoxin using and evanescent wave fiber-optic biosensor”, Applied Biochemistry and Biotechnology, 60, 189-202, 1996.
[22] H. Muramatsu, E. Tamiya, “Viscosity monitoring with a piezo- electric quartz crystal and its application to determination of endotoxin by gelation of limulus amebocyte lysate”, Analytica Chimica Acta, 215, 91-98, 1988.
[23] M. Kastowsky, T. Gutberlet, “Molecular modeling of the three- dimensional structure and conformational flexibility of bacterial lipopolysaccharide”, Journal of Bacteriology, 174, 4798-4806, 1992.
[24] U. Seydel, H. Labischinski, M. Kastowsky, “Phase behavior, supramolecular structure and molecular conformation of lipopolysaccharide”, Immunobiology, 187, 191-211, 1993.
[25] H. R. Buller, A. Sturk, J. Levin, “Bacterial endotoxins structure, biomedical significance, and detection with the limulus amebocyte lysate test.”, Alan R. liss, 3-29, 1985.
[26] S. W. Watson, J. Levin, T. J. Novitsky, “Detection of bacterial endotoxins with the limulus amebocyte lysate test”, Alan R. liss, 8-9, 1985.
[27] 楊朝欽, 壓電石英晶體上電極界面的修飾與其在生化檢測上的應用, 國立成功大學醫學工程研究所碩士論文, 1996.
[28] D. Ivnitski, A. H. Ihab, P. Atanasov, E. Wilkins, “Biosensors for detection of pathogenic bacteria.” Biosensors & Bioelectronics, 14, 599-624, 1999.
[29] A. F. Collings, F. Caruso, “Biosensors: recent advances”, Reports on Progress in Physics, 60, 1397-1445, 1997.
[30] 田蔚城, 生物技術的發展與應用, 九州圖書文物, 1997.
[3 ] A. J. Cunningham, “Introduction to bioanalytical sensors”, John Wiley & Sons, 1998.
[32] B. D. Ratner, A. S. Hoffman, F. J. Schoen, J. E. Lemons, “Biomaterials Science: An Introduction to Materials in Medicine”, 1996.
[33] A. L. Plant, “Supported hybrid bilayer membranes as Rugged cell membrane mimics”, Langmuir, 15, 5128-5135, 1999.
[34] A. J. Cunninghan, “Introduction to bioanalytical sensors”, Wiley-Interscience Publication, 1998.
[35] A. A. Karyakin, G. V. Presnova, M. Y. Rubtsova, A. M. Egorov, “Oriented immobilization of antibodies onto the gold surface via their native thiol groups”, Analytical Biochemistry , 72, 3805-3811, 2000.
[36] R. G. Nuzzo, B. R. Zegarski, L. H. Dubois, “Fundamental studies of the chemisorption of organosulfur compounds Au(111). Implications for molecular self-assembly on gold surfaces”, Journal of the American Chemical Society, 109, 733-740, 1987.
[37] M. I. Pividori, A. Merkoci, S. Alegret, “Electrochemical genosensor design: immobilization of oligonucleotides onto transducer surfaces and detection methods”, Biosensors & Bioelectronics, 15, 291-303, 2000.
[38] I. Willner., ”Enzyme-linked amplified electrochemical sensing of oligo- nucleotide-DNA interactions by means of the precipitation of an insoluble product and using impedance spectroscopy”, Langmuir, 15, 3703-3706, 1999.
[39] H. C. Yoon, M. Y. Hong, H. S. Kim, “Affinity biosensor for avidin using a double functionalized dendrimer monolayer on a gold electrode”, Analytical Biochemistry, 282, 121-128, 2000.
[40] 田昭武, 電化學研究方法, 科學出版社, 1984.
[41] D. A. Harrington, P. Driessche, “Stability and electrochemical impedance of mechanisms with a single adsorbed species”, Journal of Electroanalytical Chemistry, 501, 222-234, 2001.
[42] G. Lang, G. Inzelt, “An advanced model of the impedance of polymer film electrodes”, Electrochimica Actra, 44, 2037-2051, 1999.
[43] K. Martinusz, G. Lang, G. Inzelt, “Impedance analysis of poly(o-phenylenediamine) electrodes”, Journal of Electroanalytical Chemistry, 433, 1-8, 1997.
[44] P. Ferloni, M. Mastragostino, L. Meneghello, “Impedance analysis of electronically conducting polymers”, Electrochimical Actra, 41, 27-33, 1995.
[45] J. Ma, Y. M. Chu, J. Di, S. C. Liu, H. N. Li, J. Feng, Y. X. Ci, “An electrochemical impedance immunoanalytical method for detecting immunological interaction of human mammary tumor associated glycoprotein ant its monoclonal antibody”, Electrochemistry Communications, 1, 425-428, 1999.
[46] R. Pei, Z. Chen, E. Wang, X. Yang, “Amplification of antigen-antibody interaction based on biotin labeled protein-streptavidin network complex using impedance spectroscopy”, Biosensors & Bioelectronics, 16, 355-361, 2001.
[47] C. M. A. Brett, A. M. O. Brett, “Electrochemistry principles, methods, and applications”, Oxford Science Publications, 1996.
[48] C. K. Sullivan, G. G. Guilbault, “Commerical quartz crystal microbalances-theory and applications”, Biosensors & Bioelectronics, 14, 663-670, 1999.
[49] R. L. Bunde, E. J. Jarvi, J. J. Rosentreter, “Piezoelectric quartz crystal biosensors”, Talanta, 46, 1223-1236, 1998.
[50] A. Hengerer, J. Decker, E. Prohaska, S. Hauck, C. Koblinger, H. Wolf, “Quartz crystal microbalance(QCM) as a device for the screening of phage libraries”, Biosensors & Bioelectronics, 14, 139-144, 1999.
[51] R. C. Ebersole, M. D. Ward, “Amplified mass immunosorbent assay with a quartz crystal microbalance”, Journal of the American Chemical Society, 110, 8623-8628, 1988.
[52] J. J. Gooding, P. Erokhin, D. B. Hibbert, “Parameters important in tuning the response of monolayer enzyme electrodes fabricated using self-assembled monolayers of alkanethiols”, Biosensors & Bioelectronics, 15, 229-239, 2000.
[53] M. J. B. Wissink, R. Beernink, A. A. Poot, G. H. M. Engbers, T. Beugeling, W. G. Aken, J. Feijen, “Relation between cell density and the secretion of von Willebrand factor and prostacyclin by human umbilical vein endothelial cell”, Biomaterials, 22, 2283-2290, 2001.
[54] J. R. Scully, D. C. Silverman, M. W. Kendig, Electrochemical impedance analysis and imterpretation”, American Society for Testing and Materials, 1993.
[55] 李英儒, 交流阻抗分析法於脂多醣體檢測之應用, 國立成功大學醫學工程研究所碩士論文, 2001.
[56] H. Lee, H. Yang, Y. T. Kim, J. Kwak, “Anion transport in prussian blue films in acetonitrile and propylene carbonate solutions”, Journal of the Electrochemical Society, 147, 3801-3807, 2000.
[57] X. Cui, D. Jiang, P. Diao, J. Li, R. Tong, X. Wang, “Assessing the apparent effective thickness of alkanethiol self-assembled monolayers in different concentrations of Fe(CN)63-/ Fe(CN)64- by ac impedance spectroscopy”, Journal of Electroanalytical Chemistry, 470, 9-13, 1999.
[58] B. Lars, M. Werner, “Super-sensitivity of an solid-state fluorine sensor: mechanistic investigations”, Solid State Ionics, 132, 31-37, 2000.
[59] 楊景雯, 石英晶體微天秤的脂多醣體檢測, 國立成功大學醫學工程研究所碩士論文, 1997.
[60] Z. Wu, B. Wang, Z. Cheng, X. Yang, S. Dong, E. Wang, “A facile approach to immobilize protein for biosensor: self-assembled supported bilayer lipid membranes on glassy carbon electrode”, Biosensors & Bioelectronics, 16, 47-52, 2001.
[61] R. Zeeman, P. J. Dijkstra, M. Hendriks, P. T. Cahalan, J. Feijen, “Successive epoxy and carbodiimide cross-linking of dermal sheep collagen”, Biomaterials, 20, 921-931, 1999.
[62] J. Y. Lee, S. M. Park, “Electrochemistry of conductive polymers”, Journal of The Electrochemical Society, 147, 4189-4195, 2000.
[63] H. Hillebrandt, M. Tanaka, “Electrochemical characterization of self-assembled alkylsiloxane monolayers on Indium-Tin Oxide(ITO) semiconductor electrodes”, The Journal of Physical Chemistry B., 105, 4270-4276, 2001.
[64] P. Diao, M. Guo, R. Tong, “Characterization of defects in the formation process of self-assembled thiol monolayers by wlwctrochemical impedance spectroscopy”, Journal of Electroanalytical Chemistry, 495, 98-105, 2001.
[65] D. Sehgal, I. K. Vijay, “A method for the high efficiency of water-solution carbodiimide-mediated amidation”, Analytical Biochemistry , 218, 87-91, 1994.
[66] R. Timkovich, “Detection of the stable addition of carbodiimide to proteins”, Analytical Biochemistry , 79, 135-143, 1977.
[67] J. V. Staros, R. W. Wright, D. M. Swingle, “Enhancement by N-Hydroxysulfosuccinimide of water-soluble carboddiimide-mediated coupling reactions”, Analytical Biochemistry, 156, 220-222, 1986.
[68] L. G. Bachas, M. E. Meyerhoff, “Theoretical models for predicting the effect of bridding group recognition and conjugate substitution on hapten enzyme immunoassay dose-response curves”, Analytical Biochemistry, 156, 223-238, 1986.

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