(3.227.235.183) 您好!臺灣時間:2021/04/13 21:18
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:顏毅廣
研究生(外文):Yen Yi-Kuang
論文名稱:全反射螢光顯微技術應用於蛋白質分子之即時偵測與操控
論文名稱(外文):Real-Time Protien Monitoring and Manipulation Using Total Internal Reflection Fluorescence Microscopy
指導教授:黃榮山
指導教授(外文):Huang Long-Sun
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:應用力學研究所
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:87
中文關鍵詞:全反射螢光顯微鏡微機電技術漸逝波
外文關鍵詞:TIRFMMEMSevanescent wave
相關次數:
  • 被引用被引用:1
  • 點閱點閱:264
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:39
  • 收藏至我的研究室書目清單書目收藏:0
自從90 年代後期人類基因計劃解出大量基因體資料,以及電腦處理資訊速度大為發展,使得蛋白質可以被快速測定。蛋白質結構、功能性與其分子間的作用,都是蛋白質體研究裡相當重要的課題。
本文首次成功地觀測與分析抗原分子產生的動態行為和其與抗體分子間作用,並量測到其動態運動三度空間的位置與速度表現,此乃是藉著整合全反射螢光顯微術與微機電製程技術,進行蛋白質在物性環境改變時,。利用全反射光場以漸逝波激發螢光,使全反射螢光顯微術具有離玻璃基材150至700奈米尺寸等級以內的觀測功能,並且其影像的訊號背景比遠高於其他的顯微技術;而在橫向尺寸的功能也藉著量測1微米的螢光粒子得到了驗證。另外,配合速度每秒30張畫面的影像擷取系統,可對標定螢光之抗原分子Anti-IgG,進行即時偵測了解其從運動至與抗體鍵結時之狀態,並加以分析。
利用微機電製程技術,設計並製作整合氧化銦錫 (ITO)透明電極與PDMS微流道之生物晶片,為了達到尺寸微小化(1.6與0.8微升),且能與符合目前商用蛋白質生物晶片之規格,與蛋白質分子IgG和Anti-IgG相容。藉此在單分子偵測上,本文首次利用全反射螢光顯微鏡即時觀察到在固液相單一抗原分子Anti-IgG結合於抗體的狀態(1張/30秒);同時也觀察到分子在流體邊界層運動行為,並成功地追蹤並分析抗原分子在流體邊界層的三度空間運動軌跡(空間範圍間距:X軸 14.7微米;Y軸 1.3微米;Z軸 0.24微米)與速度表現,與流體理論作驗證,發現反應物分子(抗原)擴散受到流體邊界層所影響。此外,藉著生物晶片以施加直流電的方式操控奈米粒子,實際達到操控粒子的運動,觀測粒子的運動狀態,並發現電壓與粒子運動速度呈線性增長,將有助於未來在蛋白質分子間結合與解離作用的研究。
由於生物分子領域上仍有許多未知的現象,全反射螢光顯微術與微機電製程技術在單分子影像之觀測將扮演重要的角色。以了解生物分子在生物晶片上之表面特性與流體邊界層在晶片表面對生物分子之擴散的影響,以利未來在研究上或是奈米生物技術上之發展潛力。
The study of biomolecular recognition has been becoming crucial to provide insights into molecular genetics, design of biosensor devices, drug design, and development of targeted drug delivery systems. The in-depth understanding of biomolecular recogntion involves adsorption, interaction and desorption as well as associated manipulation between biomolecules. In this present study, we have successfully demonstrated a single biomolecular detection and real-time tracking of anti-IgG in a microchannel using total internal reflection flurorescence (TIRF) microscopy for illustration of protein adsorption and recognition.
TIRF microscopy is a well-suited technique for real-time imaging and monitoring of a single protein molecule in nano-layer fluidics due to its unique evanencent wave at the optically index-mismatch interaface that may excites fluorescences at the transparent near-wall region. Recent advances in charge coupled device (CCD) camera detection efficiency and speed have enabled the microscopy of temporal and spatial resolutions to be far-reaching 0.033 ms and 0.3 μm, respectively. A modified inverted TIRF microscopy was newly established, which allows a directed laser beam underneath through the inverted microscopy to be incident in a critical angle into the surface inside the microchannel. In this microchannel, the conductive ITO film was deposited to be electrically feedthrough for electrical manipulation. Based on the fluorescent beads analysis, which are 1.1 μm in size, the capability of TIRFM for single molecules monitoring and tracking, even the measurement in lateral size of molecules was demonstrated.
In this study, the TIRFM incorporates a MEMS-based electrical control biochip to monitor and track the motion of a single antigen molecule in which the real-time position and velocity of each frames were tracked and measured. The motion of an antigen molecule was founded to be dominated in a hydrodynamic boundary layer. A further comparison of experimental results with theoretics appears to be deviated unexpectedly, which provokes a further thought that a nano-layer fluidics exhibits a novel non-classical fluidic characteristics.
At last, the motion of fluorescence nanobeads manipulated by the application of DC voltage was real-time monitored. The velocities of beads were shown to be in a linear accordance with the applied voltages. As a result of the accordance, the manipulation of nonobeads with electrical control was demonstrated and verified.
中文摘要 1
Abstract 3
致謝 5
目錄 7
圖表目錄 10
表格目錄 13
第 1 章 緒論 14
1-1 前言 14
1-1-1 蛋白質即時影像偵測 14
1-1-2 蛋白質分子操控 15
1-1-3 全反射螢光顯微技術與微機電技術 15
1-2 研究動機 16
1-3 文獻回顧 18
1-3-1 單分子偵測 18
1-3-2 細胞運輸泡偵測 20
1-3-3 與其他技術結合應用 21
1-4 研究方法 23
1-5 章節提要 24
第 2 章 全反射螢光顯微鏡原理與架設 25
2-1 全反射螢光顯微鏡之基本原理 25
2-1-1 漸逝波[11] 26
2-1-2 螢光強度之計算 28
2-1-3 中間層 29
2-2 全反射螢光顯微鏡之光學架設 30
2-2-1 螢光染劑 31
2-2-2 整體光學架構 34
2-2-3 數值孔徑 36
2-2-4 螢光濾鏡組 39
2-2-5 螢光影像擷取系統 41
2-3 全反射螢光顯微鏡之特性與應用 43
2-3-1 穿透深度 43
2-3-2 訊號背景比 44
2-3-3 即時影像觀測 45
第 3 章 電控式生物晶片設計與製作 47
3-1 電控式生物晶片之設計理念 47
3-1-1 文獻回顧 47
3-1-2 抗原-抗體之鍵結 49
3-1-3 專一性結合作用 51
3-1-4 抗原-抗體結合作用之可逆性 52
3-1-5 動力常數 54
3-1-6 質量運送效應[25] 56
3-1-7 電控式生物晶片之設計 58
3-2 電控式生物晶片之製備方法 60
3-2-1 氧化銦錫電極之製程 60
3-2-2 微流道之製程 61
3-2-3 生物分子固定化 65
第 4 章 實驗結果與分析 68
4-1 微奈米粒子之即時偵測與分析 68
4-1-1 訊號背景比 68
4-1-2 橫向尺寸量側 71
4-2 蛋白質單分子即時偵測之定性分析 75
4-2-1 抗體與抗原結合反應之單分子偵測 75
4-2-2 微粒子操控與即時偵測 80
第 5 章 結論與未來展望 83
第 6 章 參考資料 85
[1] http://www.sciam.com.tw/read/readshow
[2] http://www.digitalgene.com.tw/bioapplication.htm
[3] Y. Harada, T. Funatsu, K. Murakami, Y. Nonoyama, A. Ishihama, and T. Yanagida, “Single-Molecule Imaging of RNA Polymerase-DNA Interactions in Real Time,” Biophys. J., vol. 76, pp. 709-715, 1999.
[4] T. Funatsu, Y. Harada, M. Tokunaga, K. Salto, and T. Yanagida,” Imaging of Single Fluorescent Molecules and Individual ATP Turnovers by Single myosin Molecules in Aqueous Solution,” Nature, vol. 374, pp. 555-559, 1995.
[5] Y. Ishii, and T. Yanagida, “Single Molecule Detection in Life Science,” Single Mol., vol.1, pp. 5-16, 2000.
[6] M. Oheim, and W. Stiihmer, “Tracking Chromaffin Granules on Their Way Through the Actin Cortex,” Eur. Biophys. J., vol. 29, pp. 67-89, 2000.
[7] J. Schmoranzer, M. Goulian, D. Axelrod, and S. M. Simon, “Imaging Constitutive Exocytosis with Total Internal Reflection Fluorescence Microscopy,” J. Cell. Bio., vol. 149, pp. 23-32, 2000.
[8] S. Nishida, Y. Funabashi, A. Ikai, “Combination of AFM with TIRFM for Nanomanipulation of Single Cells,” Ultramicroscopy, vol. 91, pp. 269-274, 2001.
[9] P. L. Edmiston, J. E. Lee, L. L. Wood, S. S. Saavedra, “Dipole Orientation Distributions in Langmuir-Blodgett Films by Planar Waveguide Linear Dichroism and Fluorescence Anisotropy,” J. Phys. Chem., pp. 775 — 784, Jan 1996.
[10] I. N. Chang, J. N. Lin, J. D. Andrade, J. N. Herron, “Orientation of Acid Pretreated Antibodies on Hydrophobic Dichlorodimethylsilane-Treated Silica Surfaces,” Journal of Colloid and Interface Science, pp. 2083 — 2089, 1995.
[11] A. Periasamy, Methods in Cellular Imaging, pp. 362-380, Oxford University press, 2001.
[12] K. Stock, R. Sailer, W. S. L. Strauss, M. Lyttek, R. Steiner, and H. Schneckenburger, “Variable-angle Total Internal Reflection Fluorescence Microscopy: Realization and Application of a Compact Illumination Device,” Journal of Microscopy, vol. 211, pp.19-29, 2003.
[13] A. B. Mathur, G. A. Truskey, and W. M. Reichert, “Atomic Force and Total Internal Reflection Fluorescence Microscopy for the Study of Force Transmission in Endothelial Cells,” Biophysical Journal. , Vol. 78, Iss. 4, pp. 1725-1736, 2000.
[14] S. Weiss, “Fluorescence Spectroscopy of Single Biomolecules,” Sceince, vol. 283, pp. 1676-1683, 1999.
[15] http://www.probes.com
[16] http://medicine.bjmu.edu.cn/department/biophysi/biophysics/LAB/NewSNL/training%20course/SPIE4098a-24.pdf
[17] M. Pluta, Advanced Light Microscopy, vol. 1, pp. 287-290, Elsevier Science press, 1988.
[18] http://www.olypusmicro.com/primer/techniques
[19] S. H. Kang, M. R. Shortreed, and E. S. Yeung, “Real-Time Dynamics of Single-DNA Molecules Undergoing Adsorption and Desorption at Liquid-Solid Interfaces,” Anal. Chem., vol. 73, pp. 1091-1099, 2001.
[20] A. N. Asanov, W. W. Wilson, and P. B. Oldham, “Regenerable Biosensor Platform: A Total Internal Reflection Fluorescence Cell with Electrochemical Control,” Anal. Chem., vol. 70, pp. 1156-1163, 1998.
[21] P. B. Oldham, and A. N. Asanov, “Control of Antibody-Antigen Binding or Dissociation by Electric Field,” Proc. SPIE, vol. 3603, pp. 156-162, 1999.
[22] Z. Liron, L. M. Tender, J. P. Golden, and F. S. Ligler, “Voltage-Induced Inhibition of Antigen-Antibody Binding at Conducting Optical Waveguides,” Biosensors and Bioelectronics, vol. 17, pp. 489-494, 2002.
[23] R. P. Buck, W. E. Hatfeid, M. Umana, and E. F. Bowden, Biosensor Technology Fundamentals and Applications, Chp. 17, pp. 219-239, Maccel Dekker, Inc., New York, 1990.
[24] http://juang.bst.ntu.edu.tw
[25] H. P. Jennissen, and T. Zumbrink, “A Novel Nanolayer Biosensor Principle,” Biosensors and Bioelectronics, vol. 19, pp. 987-997, 2004.
[26] 詹子欣, “光纖生物感知器在心臟血管疾病致病因子C-reactive Protein 之快速檢驗,” 國立陽明大學放射醫學研究所碩士論文, 2003.
[27] D. C. Duffy, J. C. McDonald, O. J. Schueller, and G. M. Whitesides, “Rapid Prototyping of Microfluidic Systems in Poly (dimethylsiloxane),” Anal. Chem., vol. 70, pp. 4974, 1998.
[28] G. Ocvirk, M. Munroe, T. Tang, R. Oleschuk, K. Westra, and D. Harrison, “Electrokinetic control of fluid flow in native poly(dimethylsiloxane) capillary electrophoresis devices,“ Electrophoresis, vol. 21, pp. 107, 2000.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
1. 吳裕益(1980)。國中高、低成就學生家庭背景及心理特質之比較研究。國立高雄師範大學教育學刊,2,161-198,高雄市。
2. 李堅萍(1996)。提昇技能教學的練習教學法。技術及職業教育雙月刊。31,40-41,台北市:教育部技術及職業教育司。
3. 李基常(1999)。技能教學創造思考教學模式之探討。泰山職訓學報。2,17-29,台北縣:行政院勞工委員會職業訓練局泰山職業訓練中心。
4. 林清財(1986)。國中學生對學校態度之調查分析。輔導月刊,22(5),31-36,台北市:中國輔導學會。
5. 林義男(1988)。國小學生家庭社經地位、父母參與、及學業成就的關係。國立台灣教育學院輔導學報,11,95-141,彰化市。
6. 秦夢群(1992)。高中教師管理心態、學生內外控與學生學習習慣與態度關係之研究。教育與心理研究,15,129-171,台北市:國立政治大學教育學系與心理學系。
7. 康自立(1994)。技藝教育之功能與做法。技藝教育,24,5-9,台北市:台北市大安高級工業職業學校。
8. 陳英豪、汪榮才、李坤崇(1993)。學習心事誰人知?國中國小學生學習適應及其相關因素之比較研究。國教之友,44(3),5-14,台南市:國立台南師範學院國教之友社。
9. 楊錦登(1999)。論述人際關係。國教輔導,38(3),48-53,台中市:國立台中師範學院。
10. 盧美貴(1982)。國小學生學習動機、態度及困擾之調查分析。教與學,4(5),23-27,台北市:台北市教育會。
11. 謝美英(1998)。青少年同儕關係與倫理。訓育研究,37 (3),55-60,台北市:中國訓育學會。
12. 羅寶鳳(1996)。成人在組織中的學習理論之初探。社教雙月刊,76,24-35,台北市:社教雙月刊雜誌。
 
系統版面圖檔 系統版面圖檔