臺灣博碩士論文加值系統

本論文為探討具有奈米解析等級的原子力顯微鏡於水產生物免疫反應快速檢測，及抗微生物胜肽與水產生物菌體之間動態反應的應用研究。本實驗之檢測晶片以玻片為基材，並於玻片上作化學處理，使其能於玻片上產生自我組裝單層（Self-Assembled Monolayers, SAMs），以便用於生物樣品之檢測。本研究首先進行石斑魚之免疫反應快速檢測，藉由抗體抗原專一性反應之檢測方式，以原子力顯微鏡對於高度變化的靈敏，由反應前後高度差偏移的現象來作為檢測之依據。此外，亦針對草蝦抗微生物胜肽與大腸桿菌、哈維氏菌及溶藻弧菌之動態反應觀察，掃描比對菌體經抗微生物胜肽處理前後之三維結構的變化，研究此胜肽之殺菌機制。實驗結果顯示，本研究成功地將原子力顯微鏡應用於水產生物研究上，達到快速檢測的目的，同時也瞭解到菌體對此抗微生物胜肽之敏感性與此胜肽之殺菌效果，對於日後研究水產生物之病菌或製藥上有很大的助益。

This thesis is to apply the atomic force microscopy (AFM) in the study of rapid diagnosis of immune response and dynamic reactions of an antimicrobial peptide (AMP) and germs for aqua cultured fish. Glass is used as the substrate, and self-assembled monolayers (SAMs) are produced on glass by chemical treatments to immobilize antibodies or germs. We first detected the specific binding between antigens and antibodies for rapid diagnosis of groupers using atomic force microscopy. We also investigated three-dimensional morphological changes of Escherichia coli, Vibrio harvyi, and Vibrio alginolyticus before and after treatments of monodoncin, an antimicrobial peptide (AMP) from tiger shrimp. Experimental results exhibit successful applications of AFM in studies of aquatic biology. AFM not only can be utilized for rapid diagnosis of immune response, but also can be used to visualize dynamic reactions of antimicrobial peptides and bacteria. It helps understand the sensitivity of bacteria to AMP and the effectiveness of AMP. As a result, AFM can be a great investigating tool in pharmaceutics for aquatic biology.

中文摘要 i
英文摘要 ii
目錄 iii
表目錄 v
圖目錄 vi
第一章 緒論 1
1-1 前言 1
1-2 文獻回顧 2
1-3 內容簡介 6
第二章 原子力顯微鏡介紹 7
2-1 原子力顯微鏡原理 7
2-2 掃描模式 8
2-2-1 接觸模式（contact mode） 10
2-2-2 非接觸模式（non-contact mode） 11
2-2-3 半接觸模式（semi-contact mode） 12
2-3 探針形貌探討 13
2-4 原子力顯微鏡於生物醫學應用之優勢 16
2-4-1 氣相掃描 19
2-4-2 液相動態掃描 21
第三章 免疫學簡介 23
3-1 免疫反應 23
3-2 抗體抗原介紹 24
3-3 抗微生物胜肽（Antimicrobial Peptides, AMP）介紹 26
第四章 檢測設備與實驗方法 29
4-1 檢測設備介紹 29
4-2 生物樣品 30
4-3 生物檢測晶片製程 30
4-4 原子力顯微鏡檢測方式 34
第五章 實驗結果與討論 36
5-1 抗體抗原免疫反應之快速檢測 36
5-2 石斑魚抗體抗原之快速檢測 39
5-2 抗微生物胜肽對菌體之動態反應 41
第六章 結論與未來展望 48
6-1 結論 48
6-2 未來展望 50
參考文獻 51
附錄 NT-MDT SFC050操作流程 60

[1] G. Binnig, H. Rohrer, C. Gerber, E. Weibel, “Vaccun tenneling”, Physica B-Condensed Matter, 109-110 (1982) 2075-2077.
[2] G. Binnig, H. Rohrer, C. Gerber, E. Weibel, “Tunneling through a controllable vacuum gap”, Applied Physics Letters, 40 (1982) 178-180.
[3] G. Binnig, H. Rohrer, “Scanning tunneling microscopy”, Helvetica Physics Acta, 55 (1982) 726-735.
[4] G. Binnig, H. Rohrer, C. Gerber, E. Weibel, ” Surface Studies by Scanning Tunneling Microscopy”, Physical Review Letters, 49 (1982) 57-61.
[5] G. Binnig, C.F. Quate, C. Gerber, “Atomic force microscopy”, Physical Review Letters, 56 (1986) 930-933.
[6] Bull, P.A., “Environmental reconstruction by electron microscopy”, Progress in Physical Geography, 5 (1984) 368-397.
[7] http://www.bioweb.com.tw/feature_content.asp?ISSID=358, 92.12.21.
[8] 邱子庭，“分子選殖與草蝦抗微生物胜肽特性之研究”，國立臺灣海洋大學水產養殖學系，碩士論文，(2001) 基隆。
[9] Tze-Ting Chiou, Jen-Leih Wu, Thomas T. Chen, Jenn-Kan Lu, “Molecular Cloning and Characterization of the cDNA of an Antimicrobial Peptide, Monodoncin, from Tiger Shrimp (Penaeus monodon)”, Marine Biotechnology, (in press), 2004.
[10] L. Reimer, “Transmission electron microscopy: Physics of Image Formation and Microanalysis”, Springer-Verlag Berlin and Heidelberg GmbH publishing, 1982.

[11] P. Grutter, D. Rugar, H.J. Mamin, “Magnetic force microscopy of magnetic materials”, Ultramicroscopy, 47 (1992) 393-399.
[12] D.W. Pohl, W. Denk, M. Lanz, “Optical stethoscopy: Image recording with resolution /20”, Applied Physics Letters, 44 (1984) 651-653.
[13] Klaus D. Jandt, “Developments and perspectives of scanning probe microscopy (SPM) on organic materials systems”, Materials Science and Engineering, R21 (1998) 221-295.
[14] 曾文聖，林良平，“原子力顯微鏡的原理及其在生物學上的應用”，科儀新知，第十九卷第六期（1998）4-15。
[15] http://elearning.stut.edu.tw/caster/3/no6/6-1.htm, 93.6.16.
[16] M.F. Tabet, F.K. Urban Ⅲ, “Comparison of atomic force microscope and rutherfod backscattering spectrometry data of nonameter size zinc islands”, Thin Solid Films, 290-291 (1996) 312-316.
[17] B. Susla, R. Czajka, W.S. Gordon, S. Szuba, J. Rauluszkiewicz, “AFM and STM investigations of a Bi2Sr2CaCu2O8 high-Tc superconductor”, Materials Science and Engineering, A217/218 (1996) 419-423.
[18] Scott S. Perry, Philip B. Merrill, “Preparation and characterization of MgO(1 0 0) surface”, Surface Science, 383 (1997) 268-276.
[19] Kaoru Sakaino, Sadao Adachi, “Study of Si(1 0 0) surface etched in TMAH solution”, Sensors and Actuators A, 88 (2001) 71-78.
[20] Patricia M. Dove, Forest M. Platt, “Compatible real-time rates of mineral dissolution by atomic force microscopy (AFM)”, Chemical Geology, 127 (1996) 331-338.
[21] Sander J. Tans, Alwin R. M. Verschueren, Cees Dekker, “Room-temperature transistor based on a single carbon nanotube”, Nature, 393 (1998) 49-52.

[22] Seunghyun Lee, Jungoh Kim, Wan Sub Shin, Ha-Jin Lee, Sunyoung Koo, Haiwon Lee, “Fabrication of nanostructures using scanning probe microscopy lithography”, Materials Science and Engineering, C24 (2004) 3-9.
[23] Z.J. Davis, G. Abadal, O. Hansen, X. Borise, N. Barniol, F. Perez-Murano, A. Boisen, “AFM lithography of aluminum for fabrication of nanomechanical systems”, Ultramicroscopy, 97 (2003) 467-472.
[24] Bernd Klehn, Sven Skaberna, Ulrich Kunze, “Wet-chemical nanoscale patterning of GaAs surface using atomic force microscopy lithography”, Superlattices and Microstructures, 25 1/2 (1999) 473-476.
[25] A.L. Weisenhorn, B. Darke, C.B. Prater, S.A.C. Gould, P.K. Hansma, F. Ohnesorge, M. Egger, S.P. Seyn, H.E. Gaub, “Immobilised proteins in buffer imaged at molecular resolution by atomic force microscopy”, Biophysical Journal, 58 (1990) 1251-1258.
[26] P. Zachee, M. Boogaerts, J. Snauwaert, L. Hellemans, “Imaging uremic red blood cells with the atomic force microscope”, American Journal of Nephrology, 14 (1994) 197-200.
[27] A. Dekker, A.A. Poot, J.A. van Mourik, M.P. Workel, T. Beugeling, A.Bantjes, J. Feijen, W.G. van Aken, “Improved adhesion and proliferation of human endothelial cells on polyethylene pre-coated with monoclonal antibodies directed against cell membrane antigens and extracellular matrix proteins”, Thrombosis and Haemostasis, 66 (1991) 715-724.
[28] C. Le Grimellec, E. Lesniewska, C. Cachia, J.P.Schreiber, F. de formal, J.P. Goudonnet, “Imaging of the membrane surface of MDCK cells by atomic force microscopy”, Biophysical Journal, 67 (1994) 36-41.

[29] L.S. Shlyakhtenko, A.A. Gall, J.J. Weimer, D.D. Hawn, Y.L. Lyubchenko, “Atomic force microscopy imaging of DNA covalently immobilished on a functionalized mica substrate”, Biophysical Journal, 77 (1999) 568-576.
[30] Kazuo Umemura, Jun Komatsu, Takayuki Uchihasashi, Nami Choi, Shukuko Ikawa, Taro Nishinaka, Takehiko Shibata, Yoshikazu Nakayama, Shinji Katsura, Akira Mizuno, Hiroshi Tokumoto, Mitsuru Ishikawa, Reiko Kuroda, “Atomic force microscopy of RecA-DNA complexes using a carbon nanotube tip”, Biochemical and Biophysical Research Communications, 281 (2001) 390-395.
[31] Tina Ide, Sven Laarmann, Lilo Greune, Hermann Schillers, Hans Oberleithner, M. Alexander Schmidt, “Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli”, Cellular Microbiology, 3 (10) (2001) 669-679.
[32] Arnaldo da Silva Jr., Omar Teschke, “Effects of the antimicrobial peptide PGLa on live Escherichia coli”, Biochemica et Biophysica Acta, 1643 (2003) 95-103.
[33] Qian Weiping, Xu Bin, Yao Danfeng, Lin Yihua, Wu Lei, Wang Chunxiao, Yu Fang, Lu Zhuhong, Wei Yu, “Site-directed immobilization of immunoglobulin G on 3-aminopropyltriethoxylsilane modified silicon wafer surface”, Materials Science and Engineering, C 8-9 (1999) 475-480.
[34] John P. Gering, Luca Quaroni, George Chumanov, “Immobilization of antibodies on glass surfaces through sugar residues”, Journal of Colloid and Interface Science, 252 (2002) 50-56.
[35] A. P. Quist, A. A. Bergman, C. T. Reimann, Seven O. Oscarsson, Bo U.R. Sundqvist, “Direct Measurement of single immunocomplex formation by atomic force microscopy”, Digital Instruments.

[36] Hong Xing You, Christopher R. Lowe, “AFM studies of protein adsorption 2. Characterization of Immunoglobulim G adsorption by detergent washing”, Journal of Colloid and Interface Science, 182 (1996) 586-601.
[37] O. Ouerghi, A. Touhami, A. Othmane, H. Ben Ouada, C. Fretigny, N. Jaffrezic-Renault, “Investigating antibody-antigen binding with atomic force microscopy”, Sensors and Actuators, B 84 (2002) 167-175.
[38] Lingyan Li, Shengfu Chen, Seajin Oh, Shaoyi Jiang, “In situ single-molecule detection of antibody-antigen binding by tapping-mode atomic force microscopy”, Analytical Chemistry, 74 (2002) 6017-6022.
[39] Martin L. Bennink, Dessy N. Nikova, Kees O. van der Werf, Jan Greve, “Dynamic imaging of single DNA-protein interactions using atomic force microscopy”, Analytica Chimica Acta, 479 (2003) 3-15.
[40] James A. Dvorak, “The application of atomic force microscopy to the study of living vertebrate cells in culture”, Methods, 29 (2003) 86-96.
[41] Hong Xing You, Joan M. Lau, Shengwen Zhang, Lei Yu, “Atomic force microscopy imaging of living cells : a preliminary study of the disruptive effect of the cantilever tip on cell morphology”, Ultramicroscopy, 85 (2000) 297-305.
[42] Yuekan Jiao, Dmitry I. Cherny, Gudrun Heim, Thomas M. Jovin, Tilman E. Schaffer, “Dynamic interactions of p53 with DNA in solution by time-lapse atomic force microscopy”, Journal of Molecular Biollogy, 314 (2001) 233-243.
[43] Marco Marra, Clara Cassinelli, “Force measurement on cell repellant and cell adhesive alginic acid coated surface”, Colloids and Surfaces B: Biointerfaces, 18 (2000) 249-259.
[44] Rajendrani Mukhopadhyay, Jan H. Hoh, “AFM force measurements on microtubeule-associated proteins: the projection domain exerts a long-range repulsive force”, FEBS Letters, 505 (2001) 374-378.
[45] Lisa M. Wilde, Stephanie Allen, Martyn C. Davies, Saul J.B.Tendler, Philip M. Williams, Clive J. Roberts, “Bifunctional atomic force microscopy probes for moleculer screening applications”, Analytica Chimica Acta, 479 (2003) 77-85.
[46] Robert B. Best, David J. Brockwell, Jose L. Toca-Herrera, Anthony W. Blake, D. Alastair Smith, Sheena E. Radford, Jane Clarke, “Force mode atomic force microscopy as a tool for protein folding studies”, Analytica Chimica Acta, 479 (2003) 87-105.
[47] Marco Morra, Clara Casinelli, Alessandra Pavesio, Davide Renier, “Atomic force microscopy evaluation of aqueous interfaces of immobilized hyaluronan”, Journal of Colloid and Interface Science, 259 (2003) 236-243.
[48] 黃艾雅，“DNA掃描探針奈米解剖技術之研究”，國立東海大學生物技術研究所，碩士論文，台中，91.1。
[49] Edda Radlein, Gunther Heinz Frischat, “Atomic force microscopy as a tool to correlate nanostructure to properties of glasses”, Journal of Non-Crystalline Solids, 222 (1997) 69-82.
[50] 田福助，“物理化學（下）”，五南圖書出版公司，90.9。
[51] 林良平，“微生物顯微鏡學”，第185-197頁，藝軒圖書出版社，台北，2002。
[52] Klaus D. Jandt, “Developments and perspectives of scanning probe microscopy (SPM) on organic materials systems”, Materials Science and Engineering, R21 (1998) 221-295.
[53] J.H. Hafner, C.-L. Cheung, A.T. Woolley, C.M. Lieber, “Structural and functional imaging with carbon nanotube AFM probes”, Progress in Biophysics & Molecular Biology, 77 (2001) 73–110.
[54] Dai, H., Hanfer, J. H., Rinzler, A. G., Colbert, D. T., Smalley, R. E., “Nanotubes as nanoprobes in scanning probe microscopy”, Nature, 384 (1996) 147-150.
[55] Jason H. Hafner, Michael J. Bronikowski, Bobak R. Azamian, Pavel Nikolaev, Andrew G. Rinzler, Daniel T. Colbert, Ken A. Smith, Richard E. Smalley, “Catalytic growth of single-wall carbon nanotubes from metal particles”, Chemical Physics Letters, 296 (1998) 195-202.
[56] Jason H. Hafner, Chin Li Cheung, Charles M. Liber, “Direct growth of single-walled carbon nanotube scanning probe microscopy tips”, Journal of the American Chemical Society, 121 (1999) 9750-9751.
[57] http://www.niea.gov.tw/analysis/publish/month/36/36th4-1.htm, 93.2.26
[58] http://140.112.78.220/~juang/ECX/Ana6.htm, 93.12.01
[59] Roitt Brostof Male etc，“IMMUNOLOGY 免疫學”， 藝軒圖書出版社， 民85.10。
[60] 郭雅音，“臨床血清免疫學”，藝軒圖書出版社，2000.9。
[61] http://www.bioweb.com.tw/feature_content.asp?ISSID=300, 93.2.26.
[62] Ivan M. Roitt, Jonathan Brostoff, David K. Male., “Immunology, 5/e”, C.V. Mosby, 1998.
[63] Charles A. Janeway, Paul Travers, Mark Walport, Mark J. Shlomchik, “Immunobiology : the immune system in health and disease, 5th ed.”, Garland Publishing, 2002.
[64] Bradshaw CM, Richard AS, Sigel MM, “IgM antibodies in fish mucus”, Proceedings of the Society for Experimental Biology and Medicine, 136(4) (1971) 1122-4.
[65] H. G. Boman, D. Hultmark, “Cell-Free Immunity in Insects”, Annual Review of Microbiology, 41 (1987) 103-126.
[66] Tossi A, Sandri L, Giangaspero A., “Amphipathic, α-helical antimicrobial peptides”, Biopolymers, 55 (2000) 4-30.

[67] Storici P. and Zanetti M., “A Novel cDNA Sequence Encoding a Pig Leukocyte Antimicrobial Peptide with a Cathelin-like Pro-sequence”, Biochemical and Biophysical Research Communications, 196 (1993) 1363-1368.
[68] Ganz T, Lehrer RI., “Antimicrobial peptides of vertebrates”, Current Opinion in Immunology, 10 (1998) 41-4.
[69] Lehrer RI, Ganz T., “Antimicrobial peptides in mammalian and insect host defence”, Current Opinion in Immunology, 11 (1999) 23-7.
[70] Ganz T, Lehrer RI., “Antimicrobial peptides of leukocytes”, Current Opinion in Hematology, 4 (1997) 53-8.
[71] Storici P, Scocchi M, Tossi A, Gennaro R, Zanetti M., “Chemical synthesis and biological activity of a novel antibacterial peptide deduced from a pig myeloid cDNA”, FEBS Letters, 337 (1994) 303-7.
[72] Verbanac D, Zanetti M, Romeo D., “Chemotactic and protease-inhibiting activities of antibiotic peptide precursors”, FEBS Letters, 317 (1993) 255-8.
[73] Andreu D, Rivas L., “Animal antimicrobial peptides: An overview”, Biopolymers, 47 (1998) 415-33.
[74] Ludtke S, He K, Huang H., “Membrane thinning caused by magainin 2”, Biochemistry, 34 (1995) 16764-9.
[75] Ludtke SJ, He K, Heller WT, Harroun TA, Yang L, Huang HW., “Membrane pores induced by magainin”, Biochemistry, 35 (1996) 13723-8.
[76] Matsuzaki K, Murase O, Fujii N, Miyajima K., “Translocation of a channel-forming antimicrobial peptide, magainin 2, across lipid bilayers by forming a pore”, Biochemistry, 34 (1995) 6521-6.

[77] Matsuzaki K, Murase O, Fujii N, Miyajima K., “An antimicrobial peptide, magainin 2, induced rapid flip-flop of phospholipids coupled with pore formation and peptide translocation”, Biochemistry, 35 (1996) 11361-8.
[78] Christensen B, Fink J, Merrifield RB, Mauzerall D., “Channel-forming properties of cecropins and related model compounds incorporated into planar lipid membranes”, Proceedings of the National Academy of Sciences USA, 85 (1988) 5072-6.
[79] Merrifield RB, Merrifield EL, Juvvadi P, Andreu D, Boman HG., “Design and synthesis of antimicrobial peptides”, Ciba Found Symp, 186 (1994) 5-26.
[80] Oren Z, Shai Y., “Mode of action of linear amphipathic alpha-helical antimicrobial peptides”, Biopolymers, 47 (1998) 451-63.
[81] Duclohier H, Molle G, Spach G., “Antimicrobial peptide magainin I from Xenopus skin forms anion-permeable channels in planar lipid bilayers”, Biophysical Journal, 56 (1989) 1017-21.
[82] Shai Y., “Molecular recognition between membrane-spanning polypeptides”, Trends in Biochemical Sciences, 50 (1995) 460-4.
[83] Cociancich S, Ghazi A, Hetru C, Hoffmann JA, Letellier L., “Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus”, Journal of Biological Chemistry, 268 (1993) 19239-45.
[84] Kragol G, Lovas S, Varadi G, Condie BA, Hoffmann R, Otvos L Jr., “The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding”, Biochemistry, 40 (2001) 3016-26.
[85] http://www.c-oneworld.com.tw/q&a/qna.htm, 93.6.3
[86] http://www.evta.gov.tw/employee/emp/001/000/a111/5-2.htm, 93.6.2.
[87] http://www.aquatwn.com.tw/publication/NO41.htm, 93.6.7.