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研究生:林玟君
研究生(外文):Wen-JiunLin
論文名稱:電化學式嗜鹼性白血球胞吐組織胺即時監測系統之開發
論文名稱(外文):Development of an Electrochemical System for Real-time Monitoring of Basophilic Histamine Exocytosis
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:生物醫學工程學系
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:49
中文關鍵詞:微孔電極嗜鹼性白血球組織胺過敏反應
外文關鍵詞:Microwell ElectrodeBasophilHistamineAllergy
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組織胺(histamine)為引起人體過敏反應的主要物質,其大部分儲存於人體內各組織的肥大細胞(mast cells)中,次要地方,例如血液中嗜鹼性白血球(basophils)及中樞神經的腦細胞。目前臨床上是以皮膚測試與特異性抗體IgE檢測為主,此固然可檢測出患者各自的過敏原,但本質性的會有其涵蓋過敏原不足,且無法即時偵測出某些藥物過敏反應,是此類檢測上的瓶頸。而過敏所引發的臨床症狀包含過敏性鼻炎、蕁麻疹、氣喘,甚至可能導致休克。因此,希望利用電化學整合微孔電極的技術,針對人類嗜鹼性白血球受到刺激後,胞吐出之組織胺進行即時偵測,藉此評估過敏反應時所引發的細胞生理。在本研究當中,首先以螢光檢測法確認懸浮型人類嗜鹼性白血球細胞株(KU-812),在鈣離子載體(calcium ionophore, A23187)的刺激下所釋放的物質為組織胺,而組織胺與o-phthalaldehyde (OPA)形成之複合物具有螢光特性,並以冷光分析儀檢測組織胺釋放量。接著,開發電化學式微孔於細胞檢測之晶片,藉由流力與細胞本身之重力的方式,將懸浮型人類嗜鹼性白血球細胞(KU-812)捕捉並安置於微孔電極(microwell electrode, MWE)中,利用安培偵測法(+0.75 V vs. Ag/AgCl)即時對細胞胞吐組織胺之電化學檢測。從螢光檢測得知,刺激30 min內分泌量約3~5 fmol。而微孔晶片實驗中,每次捕捉到1~5個細胞,刺激1~7.5 min內可觀測到一些不連續之突波電流(current spikes),其響應電流約數個至數千個pA不等,響應時間約0.6~50 sec且持續約30~195 sec。顯微鏡下觀察有突波個案者,其受測細胞均有胞吐的現象產生,將累積的突波電流之總電量(1,639 pC)進行換算,平均每個有3.2~24.6 fmol的組織胺被電極偵測到。此微孔晶片適用於量測細胞個別的差易,提供細胞良好的檢測平台,未來加入陣列式晶片之概念後,能達即時性及低成本之過敏檢測,更可評估不同類型細胞之生理狀態。
Histamine is the major substance that triggers human allergic reactions. Most of it is stored in mast cells in various tissues in the body, and some of it is stored in basophils in the blood and brain cells in the central nervous system. To determine the type of allergy, it is usually performed by skin tests or specific IgE detection. Even though these two methods can detect allergens, not all types of allergens can be detected. In addition, they cannot detect some drug allergies immediately. These would limit the applications of the two commonly used methods. The complications developed such as allergic rhinitis, urticarial, and asthma could eventually lead to shock. Therefore, we would like to use the integrated micro-electrode electrochemical technology to detect the histamine exocytosed by stimulated basophils to assess the cellular physiology in the case of an allergy. In this study, the histamine released by the cells from the suspended human basophilic leukemia cell line (KU-812) triggered by calcium ionophores, A23187, would be confirmed by using a fluorescence assay because the complex of histamine and o-phthalaldehyde (OPA) has a fluorescent property. A fluorescence analyzer can then be used to quantify the released histamine. Then, an electrochemical microchip is developed. By using the flow and gravity forces, the suspended basophil was captured and placed on the microwell electrode, MWE. Amperometry is then used for a real-time detection of the exocytosed histamine (+0.75 V, vs. Ag/AgCl). Fluorescent result showed that histamine was released about 3~5 fmol after being stimulated with calcium ionophore for 30 min. For amperometric detection of exocytosis at KU-812 cells, a non-continuous current with randomly occurring spikes that had an average area of 1,639 pC and average histamine release of 3.2~24.6 fmol per cell was observed. The signal lasted for 30 to 195 seconds. The response time was 0.6 to 50 seconds and the response current was 0.8 pA to 7.5 nA. Also, the exocytosis signals from 1~5 trapped cells appeared after the cells being stimulated for 1~7.5 min every time, due to the exocytotic activity that can be stimulated by calcium ionophore by increasing the Ca2+ influx into cytoplasm. This microchip can be used to differentiate the cells and provide a detection platform. After the integration of this chip with microarrays, allergen detection can be made real-time and has a low cost. It can also be applied to cellular physiological simulation and assessment for cells.
Abstract...................................................I
摘要......................................................II
致謝.....................................................III
Contents..................................................IV
List of Figures...........................................VI
List of Tables..........................................VIII
Chapter1. Introduction...............................1
1.1 Allergic Reaction..................................1
1.2 Method of Clinical Diagnosis of Allergy............4
1.3 Transport of Intracellular Calcium Ionophore Exocytosis.................................................5
1.4 The Importance and Methods of Histamine Detection..9
1.5 Microelectrochemistry Used for Cell Secretion Detection.................................................12
1.6 The Manipulation of Cell..........................15
1.7 The Aim of This Study.............................18
Chapter2. Materials and Experiments.................20
2.1 Equipments........................................20
2.2 Materials.........................................20
2.2.1 Chemicals for Microfabrication....................20
2.2.2 Reagents for Cell Culture.........................21
2.2.3 Reagents for Sensitization of Cells...............21
2.2.4 Reagents for Fluorescence Detection...............21
2.2.5 Reagents for Electrochemical Detection............21
2.2.6 Test solution.....................................22
2.3 Cell Line and Cell Culture........................22
2.4 Fluorescence Detection for the Basophil...........22
2.5 Fabrication and Integration of the MWE Chip.......23
2.5.1 Chip Fabrication..................................23
2.5.2 An Appropriate Photoresist Select.................25
2.6 Electrochemical Characteristics and Function of MWE.......................................................25
2.6.1 Validation of MWE Characteristics.................25
2.6.2 Cells Manipulation................................26
2.7 Real-time Detection of Exocytosis from KU-812.....26
2.8 Framework of the System...........................27
2.8.1 Fluorescence Detection............................27
2.8.2 Electrochemical Detection.........................28
Chapter3. Results and Discussion....................29
3.1 Fluorescence Detection for the Basophil...........29
3.2 Photoresist on MWE by Using Electrochemical Deposition................................................31
3.3 Electrochemical Characteristics of the MWE........32
3.4 Entrapment of Cell inside MWE.....................34
3.5 Real-time Detection of Exocytosis from KU-812.....35
Chapter4. Conclusion................................43
References................................................45

[1] K. Tasaka, New advances in histamine research, 1st ed., Tokyo, Springer Verlag, New York: 1994.
[2] P. Gell, Coombs RRA, eds. Clinical Aspects of Immunology. 1st ed. Oxford, England: Blackwell, 1963.
[3] R. Lewis, Life, 3rd ed., McGraw Hill, New York: 1998.
[4] T. V. Rajan, The Gell-Coombs classification of hypersensitivity reactions a re-interpretation, Trends Immunol, 2003, 24, 376-379.
[5] C. Prussin, D. D. Metcalfe, IgE, mast cells, basophils, and eosinophils, J Allergy Clin Immunol, 2003, 111, 486-494.
[6] A. B. Kay, Allergy and Allergic Disease. Blackwell Science, Oxford, 1967.
[7] K. Ishizaka, T. Ishizaka, Identification of gamma-E antibodies as a carrier of reaginic antibody, J. Immunol, 1967, 99, 1187-1198.
[8] D. M. Segal, J. D. Taurog, and H. Metzger, Dimeric immunoglobulin E serves as a unit signal for mast cell degranulation. Proc. Natl. Acad. Sci. USA, 1977, 74, 2993-2997.
[9] T. Bieber. FcεRI-expressing antigen-presenting cells: new players in the atopicgame, J. Immunol, 1997, 18, 311-313.
[10] L. Larson, J. B., and G. J. Gleich, Maximal rise in IgE antibody following ragweed pollination season, J. Allergy Clin. Immunol, 1975, 55, 10-15.
[11] http://fig.cox.miami.edu/~lfarmer/BIL265/immunesystem.htm.
[12] R. Hiller, S. Laffer, C. Harwanegg, M. Huber, W. M. Schmidt, A. Twardosz, B. Barletta, W. M. Becker, K. Blaser, H. Breiteneder, M. Chapman, R. Crameri, M. Duchêne, F. Ferreira, H. Fiebig, K. H. Sommergruber, T. P. King, T. K. Janke, V. P. Kurup, S. B. Lehrer, J. Lidholm, U. Müller, C. Pini, G. Reese, O. Scheiner, A. Scheynius, H. D. Shen, S. Spitzauer, R. Suck, I. Swoboda, W. Thomas, R. Tinghino, M. V. H. Hamsten, T. Virtanen, D. Kraft, M. W. Müller, R. Valenta, Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment, The FASEB Journal, 2002, 16, 414-416.
[13] J. Lin, P. R. Shewry, D. B. Archer, K. Beyer, B. Niggemann, H. Haas, P. Wilson, M. J.C. Alcocer, The potential allergenicity of two 2S albumins from soybean (glycine max): a protein microarray approach, Int Arch Allergy Immunol, 2006, 141, 91-102.
[14] J. Rivera, J. R. Cordero, Y. Furumoto, Macromolecular protein signaling complexes and mast cell responses: a view of the organization of IgE-dependent mast cell signaling, Mol Immunol, 2001, 38, 1253-1258.
[15] C. Tkaczyk, A. M. Gilfillan, FcRI-dependent signaling pathways in human mast cells, Clin Immunol, 2001, 99, 198-210D. Lawson, C. Fewtrell, B. Gomperts, M. C. Raff, Anti-immunoglobulin-induced histamine secretion by rat peritoneal mast cells studied by immunoferritin electron microscopy, J Exp Med, 1975, 142, 391-402.
[16]D. Lawson, C. Fewtrell, B. Gomperts, M. C. Raff, Anti-immunoglobulin-induced histamine secretion by rat peritoneal mast cells studied by immunoferritin electron microscopy, J Exp Med, 1975, 142, 391-402.
[17] J. W. Deitmer, A. J. Verkhratsky, C. Lohr, Calcium signaling in glial cells, Cell Calcium, 1998, 24, 405-416.
[18] R. D. Burgoyne, M. J. Clague, Clacium and calmodulin in membrane fusion, Biochim Biophys Acta, 2003, 1641, 137-143.
[19] M. Hoth, R. Penner, Depletion of intracellular calcium stores activates a calcium current in mast cells, Nature, 1992, 355, 353-356.
[20] T. T. Ching, A. L. Hsu, A. J. Johnson, Phosphoinositide 3-kinase facilitates antigen-stimulated Ca2+ influx in RBL-2H3 mast cells via a phosphatidylinositol 3,4,5-trisphosphate-sensitive Ca2+ entry mechanism, J Biol Chem, 2001, 276, 14814-14820.
[21] P. Rohlich, P. Anderson, B. J. Uvnas, Electron microscope observations on compound 48/80-induced degranulation in rat mast cells, J Cell Biol, 1971, 51, 465-483.
[22] N. C. Moran, B. Uvnas, B. Westerholm, Release of 5-HT and histamine from mast cells, Acta Physiol Scand, 1962, 56, 26-41.
[23] J. C. Foreman, J. L. Mongar, B. D. Gomperts, Calcium ionophores and movement of calcium ions following the physiological stimulus to a secretory process, Nature, 1973, 245, 249-251.
[24] B. J. Abbott, D. S. Fukuda, D. E. Dorman, J. L. Occolowitz, M Debona, L. Farhner, Microbial transformation of A23187, a divalent cation ionophore antibiotic, Antimicrob Agents Chemother, 1979, 16, 808-816.
[25] R. Heidelberger, G. Mattews, Inhibition of calcium influx and calcium current by gamma-aminobutyric acid in single synaptic terminals, Proc Nati Acad Sci, 1991, 88, 7135-7139.
[26] I. M. Roitt, J. Brostoff, D. K. Male., Immunology, 2nd ed., Mosby, London, Gower, New York: 1989.
[27] G. Marone, Human basophils and mast cells: clinical aspects, 1st ed., Basel, Karger, New York: 1995.
[28] L. Maintz, N. Novak, Histamine and histamine intolerance, Am J Clin Nutr, 2007, 85, 1185-1196.
[29] M. A. Kaliner, D. D. Metcalfe,The mast cell in health and disease, 1st ed., MarcelDekker Inc., New York: 1992.
[30] D. Lagunoff, E. Y. Chi, Mast cell secretion: membrane events, J Invest Dermat, 1978, 71, 81-84.
[31] K. L. Barrell, F. L. Pearce, A comparison of histamine secretion from isolated peritoneal mast cells of the mouse and rat, Int Archs Allergy appl Immun, 1983, 71, 234-238.
[32] D. L. Walters, J. E. James, F. B. Vest, H. T. Karnes, A comparison of fluorescence versus chemiluminescence detection for analysis of the fluorescamine derivative of histamine by HPLC, Biomed Chromatogr, 1994, 5, 207-211.
[33] T. Yoshitake, F. Ichinose, H. Yoshida, K. Todoroki, J Kehr, O. Inoue, H. Nohta, M. Yamaguchi, A sensitive and selective determination method of histamine by HPLC with intramolecular excimer-forming derivatization and fluorescence detection, Biomed Chromatogr, 2003, 17, 509-516.
[34] K. Plhel, S. Hsieh, J. W. Jorgenson, R. M. Wightman, Electrochemical detection of histamine and 5-hydroxytryptamine at isolated mast cells, Anal Chem, 1995, 67, 4514-4521.
[35] T. Katsu, H. Hirodo, Determination of histamine release from mast cells using a histamine-sensitive membrane electrode, Anal Chim Acta, 1999, 396, 189-193.
[36] H. Morita, M. Konishi, Electrogenerated chemiluminescence derivatization reagents for carboxylic acids and amine in high-performanceliquid chromatography using tris (2, 2-bipyridine) ruthenium (II), Anal Chem, 2002, 74, 1584-1588.
[37] S. A. Raspopov, A. E. Faramawy, B. A. Thomson, K. W. M. Siu, Infrared multiphoton dissociation in quadrupole time-of-flight mass spectrometry: top-down characterization of proteins, Anal Chem, 2006, 78, 4572-4577.
[38] L. M. Lichtenstein, P. S. Norman, J. T. Connell, Comparison between skin-sensitizing antibody titers and leukocyte sensitivity measurements as an index of the severity of ragweed hay fever, J Allergy, 1967, 40, 160-167.
[39] P. A. Shore, A. Burkhalter, V. H. Cohn, A method for the fluorometric assay of histamine in tissues, J Pharmacol Exp Ther January, 1959, 127, 182-186.
[40] L. Juhlin, W. B. Shelley, Detection of histamine by a new fluorescent o-phthalaldehyde stain, J Histochem Cytochem, 1966, 14, 525-528.
[41] Z. Z. Zhao, P. B. Sugerman, L. J. Walsh, N. W. Savage, A fluorometric microassay for histamine release from human gingival mast cells, J Periodont Res, 2001, 36, 233-236.
[42] P. E. Marszalek, B. Farrell, P. Verdugo, and J. M. Fernandez, Kinetics of release of serotonin from isolated secretory granules. I. Amperometric detection of serotonin from electroporated granules. Biophys J, 1997, 73, 1160-1168..
[43] D. J. Michael, R. M. Wightman, Electrochemical monitoring of biogenic amine neurotransmission in real time, J Pharm Biomed Anal, 1999, 19, 33-46.
[44] C. L. Murrant, M. B. Reid, Detection of reactive oxygen and reactive nitrogen species in skeletal muscle, Microsc Res Tech, 2001, 55, 236-248.
[45] P. Y. Chiou, A. T. Ohta, M. C. Wu, Massively parallel manipulation of single cells and microparticles using optical images, Nature, 2005, 436, 370-372.
[46] C. S., A. H., S. P., J. E., T. R., and R. T., Fully automated microchip system for the detection of quantal exocytosis from single and small ensembles of cells, Lab Chip, 2008, 8, 323-329.
[47] P. Chen, B. Xu, N. Tokranova, X. Feng, J. Castracane, and K. D. Gillis, Amperometric detection of quantal catecholamine secretion from individual cells on micromachined silicon chips, Anal Chem 2003, 75, 518-524.
[48] C. Amatore, S. Arbault, C. Bouton, J. C. Drapier, H. Ghandour, and A. C. Koh, Real-time amperometric analysis of reactive oxygen and nitrogen species released by single immunostimulated macrophages, Chembiochem 2008, 9, 1472-1480.
[49] R. T. Kennedy, L. Huang, M. A. Atkinson, and P. Dush, Amperometric monitoring of chemical secretions from individual pancreatic beta-cells, Anal Chem 1993, 65, 1882-1887.
[50] J. M. Finnegan, K. Pihel, P. S. Cahill, L. Huang, S. E. Zerby, A. G. Ewing, R. T. Kennedy, and R. M. Wightman, Vesicular quantal size measured by amperometry at chromaffin, mast, pheochromocytoma, and pancreatic beta-cells, J Neurochem, 1996, 66, 1914-1923.
[51] J. Voldman, Engineered systems for the physical manipulation of single cells, Curr Opin Biotechnol, 2006, 17, 532-537.
[52] C. E. Sims, N. L. Allbritton, Analysis of single mammalian cells on-chip, Lab Chip, 2007, 7, 423-440.
[53] J. E. Ali, P. K. Sorger, K. F. Jensen, 0Nature, 2006, 442, 403-411.
[54] M. Deutsch, A. Deutsch, O. Shirihai, I. Hurevich, E. Afrimzon, Y. Shafran, N. Zurgil, A novel miniature cell retainer for correlative high-content analysis of individual untethered non-adherent cells, Lab Chip, 2006, 6, 995-1000.
[55] B. M. Taff, J. Voldman, A scalable addressable positive-dielectrophoretic cell-sorting array, Anal Chem, 2005, 77, 7976-7983.
[56] C. Y. Chang, Y. Takahashi, T. Murata, H. Shiku, H. C. Chang, T. Matsue, Entrapment and measurement of a biologically functionalized microbead with a microwell electrode, Lab Chip, 2009, 9, 1185-1192.
[57] D. L. Walters, J. E. James, F. B. Vest, H. T. Karnes, A comparison of fluorescence versus chemiluminescence detection for analysis of the fluorescamine derivative of histamine by HPLC, Biomed Chromatogr, 1994, 5, 207-211
[58] A. J. Bard, L. R. Faulkner, Electrochemical methods fundamentals and applications, 2nd ed., Chichester, John Wiley, Hoboken, NJ: 2001
[59] D. W. M. Arrigan, Nanoelectrodes, nanoelectrode arrays and their applications, Analyst, 2004, 129, 1157-1165
[60] 賴冠宇, 以電化學式微流體晶片之胞吐組織胺的線上監測, 國立成功大學/醫學工程研究所碩士論文, 2010.
[61] 張景裕, 電化學式單細胞分泌即時監測系統之開發, 國立成功大學/醫學工程研究所博士論文, 2007

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