進階搜尋


 
系統識別號 U0026-0812200913595040
論文名稱(中文) 結合銀奈米粒子標記與銀訊號增強技術搭配光學與電阻式晶片於微免疫分析之研究
論文名稱(英文) Development of an Immunoassay Based on Grayscale and Impedance Measurements Utilizing Nanoparticle with Signal Amplification Techniques
校院名稱 成功大學
系所名稱(中) 工程科學系碩博士班
系所名稱(英) Department of Engineering Science
學年度 95
學期 2
出版年 96
研究生(中文) 陳威廷
研究生(英文) Wei-ting Chen
電子信箱 n9694156@mail.ncku.edu.tw
學號 n9694156
學位類別 碩士
語文別 中文
論文頁數 118頁
口試委員 口試委員-張長泉
口試委員-林弘萍
指導教授-林裕城
中文關鍵字 銀析出現象  微機電  酵素連結免疫吸附分析  生物晶片  銀奈米粒子 
英文關鍵字 immunoassay  silver precipitation  nanoparticles  biochip  MEMS 
學科別分類
中文摘要 本研究之設計概念與實驗執行在於利用微生物晶片技術,結合銀奈米粒子並配合金屬銀析出之反應,且以光學式與電阻式偵測系統來量測免疫反應訊號,建立起新型之免疫分析系統,提供另一種有效且方便的免疫分析檢測方式。美國根據國家院內感染監測系統統計,院內感染病人身上培養出的菌株,金黃色葡萄球菌佔13%,金黃色葡萄球菌是造成嚴重感染菌血症之重要病原菌,而且近年來有逐漸增加之趨勢。所以在抗原抗體標定的選取上,利用金黃色葡萄球菌特有的protein A與免疫球蛋白G (IgG) 作為免疫分析模型,以探討新型免疫分析檢驗之可行性與效能。此外,在微晶片上執行免疫分析反應具有改良多樣性、低樣本用量與高靈敏度等特性,且可以縮短偵測反應的時間。實驗的架構朝向臨床上廣泛使用的三明治免疫分析模式,利用上下兩層已知抗體來對中間待測抗原作定性與定量之分析。有別於酵素連結免疫吸附分析(enzyme-linked immunosorbent assay, ELISA),我們採用現行抗原抗體在晶片表面之固定技術,並加入銀析出增強溶液,來增顯免疫的反應偵測訊號。利用光學與電性式偵測結果顯示,本研究方法已可完成免疫分析的步驟與目標,光學與電性偵測靈敏度分別達到10-9 g/mL (1 ng/mL)與10-10 g/mL (0.1 ng/mL),且使用的偵測方式也可搭配銀析出現象產生可供檢測之訊號,大幅縮短檢驗時間為30分鐘內,比傳統ELISA檢驗所需3到4小時,更為快速。此外,利用微機電技術、奈米粒子標記技術與銀訊號放大增強方法,建立一個全新、快速且靈敏之免疫分析系統,相信本研究之建立,未來將對臨床免疫分析方法提供嶄新的觀念與貢獻。
英文摘要 In this study, silver nanoparticle-labeled antibody is coupled with silver enhancement method and microchip to provide an effective and convenient immunoassay. Besides, the immunoassay has the advantages of versatile applications, lower sample consumption, high sensitivity and shorter detection time. According to the survey of infection system, Staphylococcus aureus is the main factor to lead to bacteremia. The application of immune analysis is widespread in biomedical field, because it is sensitivity and specificity. The design and experiment of this research lie in combining the biochip and nanoparticles to set up a new immunoassay system. In this study, Protein A from S. aureus and immunoglobulin G (IgG) are selected as the model immunoassay to estimate the feasibility and efficiency of the novel immunoassay. There is major format developed in this study, sandwich immunoassay (three-layer format), primary and secondary antibodies are used to quantify and qualify test antigens. Compared to traditional enzyme-linked immunosorbent assay (ELISA), we adopt the immobilization of antigen or antibody on the chip surface, and introduce silver enhancement method to amplify the detection signal generated by the silver nanoparticles. The experimental data of optical and electric detection show that the silver-precipitation immunoassay can be worked. The relationship between sample concentration and detection signal is discussed and the detection limit (sandwich assay) for the sample antigen are 10-9 g/mL (1 ng/mL) and 10-10 g/mL (0.1 ng/mL) in optical and electric system respectively. Further, the detection time of our system is 30 minutes shorter than 3 to 4 days of transitional ELISA method. In addition, we combine MEMS technology and silver enhancement to achieve a novel immunoassay system. The high applicability and biochemical efficiency of this study can provide an alternative for rapid, sensitive and convenient immunoassay.
論文目次 中文摘要 I
Abstract III
誌謝 V
目錄 VI
圖目錄 X
表目錄 XV
第一章 序論 1
1-1 研究背景 1
1-2 文獻回顧 4
1-2-1 免疫分析基本理論 4
1-2-2 免疫分析法之發展 5
1-2-3 傳統酵素聯結免疫吸附分析法─ELISA 14
1-3 研究目的與動機 19
1-4 實驗架構 21
1-5 研究策略 22
第二章 材料與方法 24
2-1 實驗藥品介紹 24
2-2 實驗材料製備與方法 25
2-2-1 銀奈米粒子合成技術 25
2-2-2 銀奈米粒子與抗體接合之原理 27
2-2-3 銀奈米粒子與抗體接合之方法 29
2-3 金屬銀析出之反應原理 31
2-4 實驗偵測儀器介紹與原理 33
2-4-1 紫外光─可見光吸收光譜儀 33
2-4-2 光學式免疫偵測平台之建立與操作歩驟 35
2-4-3 電阻式免疫偵測平台之建立與操作歩驟 40
第三章 免疫分析晶片之設計與製作 42
3-1 晶片結構與設計 42
3-1-1 光學掃描晶片 42
3-1-2 電性阻抗晶片 43
3-2 光罩與設計 46
3-3 晶片製程流程 48
3-3-1 基材清洗 49
3-3-2 金屬蒸鍍 52
3-3-3 微影製程 55
3-3-4 金屬層蝕刻 59
3-3-5 PDMS免疫反應區製作 62
3-3-6 晶片滅菌 65
第四章 銀析出免疫分析模型之結果與討論 67
4-1 決定基材 68
4-2 銀增強溶液測試 69
4-2-1 銀奈米粒子於銀增強溶液之灰階值測試 71
4-2-2 銀奈米粒子於銀增強溶液之電性測試 73
4-3 銀奈米粒子與抗體接合之結果 75
4-4 銀奈米粒子與抗體接合後之催化能力驗證 78
4-4-1 銀奈米粒子與抗體接合後之灰階值實驗 78
4-4-2 銀奈米粒子與抗體接合之電性實驗 81
4-5 光學掃描式銀析出免疫分析實驗 83
4-5-1 光學掃描式銀析出免疫分析步驟 83
4-5-2 銀增強溶液反應時間之影響 86
4-5-3 待測抗原濃度對應灰階值之實驗與結果 87
4-6 電性阻抗式銀析出免疫分析實驗 91
4-6-1 微電極免疫分析晶片 92
4-6-2 電性阻抗式銀析出免疫實驗步驟 93
4-6-3 待測抗原濃度對應阻抗值之實驗與結果 95
第五章 結論與建議 102
5-1 結論. 102
5-2 建議. 106
5-3 未來展望 107
參考文獻 108
參考文獻 [1] E. Engvall and P. Perlmann, “Enzyme-linked immunosorbent assay, Elisa. 3. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes,” Journal of Immunology, 109, 129, 1972.
[2] G. Sakai, K. Ogata, T. Uda, N. Miura and N. Yamazoe, “A surface plasmon resonance-based immunosensor for highly sensitive detection of morphine,” Sensors and Actuators B, 49, 5, 1998.
[3] B. K. Oh, Y. K. Kim, W. Lee, Y. M. Bae, W. H. Lee and J. W. Choi, “Immunosensor for detection of legionella pneumophila using surface plasmon resonance,” Biosensors and Bioelectronics, 18, 605, 2003.
[4] G. U. Lee, D. A. Kidwell and R. J. Colton, “Sensing discrete streptavidin-biotin interactions with atomic force microscopy,” Langmuir, 10, 354, 1994.
[5] L. Li, S. Chen, S. Oh and S. Jiang, “In situ single-molecule detection of antibody-antigen binding by tapping-mode atomic force microscopy,” Analytical Chemistry, 74, 6017, 2002.
[6] S. H. Lee, D. D. Stubbs, J. Cairney and W. D. Hunt, “Rapid detection of bacterial spores using a quartz crystal microbalance (QCM) immunoassay,” IEEE Sensors Journal, 5, 737, 2005.
[7] H. Zeng, H. Wang, F. P. Chen, H. Xin, G. Wang, L. Xiao, K. Song, D. Wu, Q. He and G. Shen, “Development of quartz-crystal-microbalance-based immunosensor array for clinical immunophenotyping of acute leukemias,” Analytical Biochemistry, 351, 69, 2006.
[8] S. Schluecker, B. Kuestner, A. Punge, R. Bonfig, A. Marx and P. Stroebel, “Immuno-Raman microspectroscopy: in situ detection of antigens in tissue specimens by surface-enhanced Raman scattering,” Journal of Raman Spectroscopy, 37, 719, 2006.
[9] D. A. Stuart, A. J. Haes, C. R. Yonzon, E. M. Hicks and R. P. Van Duyne, “Biological applications of localised surface plasmonic phenomenae,” IEE Proceeding Nanobiotechnology, 152, 13, 2005.
[10] J. P. Gosling, “A decade of development in immunoassay methodology,” Clinical Chemistry, 36, 1408, 1990.
[11] J. S. Rossier and H. H. Girault, “Enzyme linked immunosorbent assay on a microchip with electrochemical detection,” Lab on a chip, 1, 153, 2001.
[12] W. C. W. Chan and S. M. Nie, “Quantum dot bioconjugates for ultrasensitive nonisotopic detection,” Science, 281, 2016, 1998.
[13] M. Bruchez, M. Moronne, P. Gin, S. Weiss and A. P. Alivisatos, “Semiconductor nanocrystals as fluorescent biological labels,” Science, 281, 2013, 1998.
[14] J. Yakovleva, R. Davidsson, A. Lobanova, M. Bengtsson, S. Eremin, T. Laurell and J. Emnéus, “Microfluidic enzyme immunoassay using silicon microchip with immobilized antibodies and chemiluminescence detection,” Analytical Chemistry, 74, 2994, 2002.
[15] C. H. Yoon, J. H. Cho, H. I. Oh, M. J. Kim, C. W. Lee, J. W. Choi and S. H. Paek, “Development of a membrane strip immunosensor utilizing ruthenium as an electro-chemiluminescent signal generator,” Biosensors and Bioelectronics, 19, 289, 2003.
[16] C. A. Mirkin, R. L. Letsinger, R. C. Mucic and J. J. Storhoff, “A DNA-based method for rationally assembling nanoparticles into macroscopic materials,” Nature, 382, 607, 1996.
[17] H. Kimura, S. Matsuzawa, C. Y. Tu, T. Kitamori and T. Sawada, “Ultrasensitive heterogeneous immunoassay using photothermal deflection spectroscopy. 2. quantitation of ultratrace carcinoembryonic antigen in human sera,” Analytical Chemistry, 68, 3063, 1996.
[18] R. Elghanian, J. J. Storhoff, R. C. Mucic, R. L. Letsinger and C. A. Mirkin, “Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles,” Science, 277, 1078, 1997.
[19] S. Brakmann, “DNA-based barcodes, nanoparticles, and nanostructures for the ultrasensitive detection and quantification of proteins,” Angewandte Chemie-International Edition, 43, 5730, 2004.
[20] J. M. Nam, C. S. Thaxton and C. A. Mirkin, “Nanoparticle-based bio–bar codes for the ultrasensitive detection of proteins,” Science, 301, 1884, 2003.
[21] X. Chu, X. Fu, K. Chen, G. L. Shen and R. Q. Yu, “An electrochemical stripping metalloimmunoassay based on silver-enhanced gold nanoparticle label,” Biosensors and Bioelectronics, 20, 1805, 2005.
[22] Y. P. Bao, T. F. Wei, P. A. Lefebvre, H. An, L. He, G. T. Kunkel and U. R. Muller, “Detection of protein analytes via nanoparticle-based bio bar code technology,” Analytical Chemistry, 78, 2055, 2006.
[23] C. Grüttner, K. Müller, J. Teller, F. Westphal, A. Foreman and R. Ivkov, “Synthesis and antibody conjugation of magnetic nanoparticles with improved specific power absorption rates for alternating magnetic field cancer therapy,” Journal of Magnetism and Magnetic Materials, 311, 181, 2007.
[24] N. T. K. Thanh and Z. Rosenzweig, “Development of an aggregation-based immunoassay for anti-Protein A using gold nanoparticles,” Analytical Chemistry, 74, 1624, 2002.
[25] S. Cobbe, S. Connolly, D. Ryan, L. Nagle, R. Eritja and D. Fitzmaurice, “DNA-controlled assembly of protein-modified gold nanocrystals,” Journal of Physical Chemistry B, 107, 470, 2003.
[26] N. L. Binnun, A. B. Lindner, O. Zik, Z. Eshhar and E. Moses, “Quantitative detection of protein arrays,” Analytical Chemistry, 75, 1436, 2003.
[27] Z. F. Ma and S. F. Sui, “Naked-eye sensitive detection of immunoglubulin G by enlargement of Au nanoparticles in vitro,” Angewandte Chemie-International Edition, 41, 2176, 2002.
[28] L. R. Hirsch, J. B. Jackson, A. Lee, N. J. Halas and J. L. West, “A whole blood immunoassay using gold nanoshells,” Analytical Chemistry, 75, 2377, 2003.
[29] M. Cais, S. Dani, Y. Eden, O. Gandolfi, M. Horn, E. E. Isaacs, Y. Josephy, Y. Saar, E. Slovin and L. Snarskt, “Metalloimmunoassay,” Nature, 270, 534, 1977.
[30] C. S. Holgate, P. Jackson, P. N. Cowen and C. C. Bird, “Immunogold-silver staining: new method of immunostaining with enhanced sensitivity,” The Journal of Histochemistry and Cytochemistry, 31, 938, 1983.
[31] M. Moeremans, G. Daneels, A. V. Dijck, G. Langanger and J. D. Mey, “Sensitive visualization of antigen–antibody reactions in dot and blot immune overlay assays with immunogold and immunogold/silver staining,” Journal of immunological methods, 74, 353, 1984.
[32] P. M. Lackie, “Immunogold silver staining for light microscopy,” Histochemistry and Cell Biology, 106, 9, 1996.
[33] M. A. Hayat (Ed.), Colloidal gold: principles, methods, and applications, 1, 252, Academic Press, New York, 1989.
[34] G. C. Manara, C. Ferrari, C. Torresani, P. Sansoni and G. D. Panfilis, “The immunogold-silver staining approach in the study of lymphocyte subporulatiins in transmission electron-microscopy,” Journal of Immunological Methods, 128, 59, 1990.
[35] T. A. Taton, C. A. Mirkin and R. L. Letsinger, “Scanometric DNA array detection with nanoparticle probes,” Science, 289, 1757, 2000.
[36] S. J. Park, T. A. Taton and C. A. Mirkin, “Array-based electrical detection of DNA with nanoparticle probes,” Science, 295, 1503, 2002.
[37] I. Alexandre, S. Hamels, S. Dufour, J. Collet, N. Zammatteo, F. D. Longueville, J. L. Gala and J. Remacle, “Colorimetric silver detection of DNA microarrays,” Analytical Biochemistry, 295, 1, 2001.
[38] Z. P. Li, C. H. Liu, Y. S. Fan, Y. C. Wang and X. R. Duan, “A chemiluminescent metalloimmunoassay based on silver deposition on colloidal gold labels,” Analytical Biochemistry, 359, 247, 2006.
[39] I. Bronstein, J. C. Voyta, G. H. G. Thorpe, L. J. Kricka and G. Armstrong, “Chemiluminescent assay of alkaline phosphatase applied in an ultrasensitive enzyme immunoassay of thyrotropin,” Clinical Chemistry, 35, 1441, 1989.
[40] E. Ishikawa, S. Hashida, T. Kohno and K. Hirota, “Ultrasensitive enzyme immunoassay,” Clinica Chimica Acta, 194, 51, 1990.
[41] A. Henglein and M. Giersig, “Formation of colloidal silver nanoparticles: capping action of citrate,” Journal of Physical Chemistry B, 103, 9533, 1999.
[42] K. A. Bogle, S. D. Dhole and V. N. Bhoraskar, “Silver nanoparticles: synthesis and size control by electron irradiation,” Nanotechnology, 17, 3204, 2006.
[43] A. Forsgren, and J. Sjöquist, ““Protein A” from S. Aureus: I. pseudo-immune reaction with human γ-globulin,” The Journal of Immunology, 97, 822, 1966.
[44] H. Ai, M. Fang, S. A. Jones and Y. M. Lvov, “Electrostatic layer-by-layer nanoassembly on biological microtemplates: platelets,” Biomacromolecules, 3, 560, 2002.
[45] K. C. Ho, P. J. Tsai, Y. S. Lin and Y. C. Chen, “Using biofunctionalized nanoparticles to probe pathogenic bacteria,” Analytical Chemistry, 76, 7162, 2004.
[46] M. Horisberger and J. Rosset, “Colloidal gold, a useful marker for transmission and scanning electron microscopy,” Journal of Histochemistry and Cytochemistry, 25, 295, 1977.
[47] C. H. Teng, K. C. Ho, Y. S. Lin and Y. C. Chen, “Gold nanoparticles as selective and concentrating probes for samples in MALDI MS analysis,” Analytical Chemistry, 76, 4337, 2004.
[48] B. M. Sergeev, M. V. Kiryukhin, M. Yu. Rubtsova and A. N. Prusov, “Synthesis of protein A conjugates with silver nanoparticles,” Colloid Journal, 65, 636, 2003.
[49] http://www.deltaunited.com.tw/twindex.htm.
[50] T. Kotnik and D. Miklavcic, “Analytical description of transmembrane voltage induced by electric fields on spheroidal cells,” Journal of Biophysical, 79, 670, 2000.
[51] S. M. Sze, VLSI Technology, 386, McGraw-Hill, New York, 1998.
[52] 莊達人,VLSI 製造技術,高立圖書,235,民國九十六年
[53] B. H. Jo, L. M. V. Lerberghe, K. M. Motsegood and D. J. Beebe, “Three dimensional nicro-channel gabrication in polydimethylsiloxane (PDMS) elastomer,” Journal of Micro- Electromechanical Systems, 9, 76, 2000.
[54] http://sms.kaist.ac.kr/~ischoi/bk/lecture/class%2009.pdf.
[55] M. A. Hayat (Ed.), Colloidal Gold: Principles, Methods, and Applications, 1, 15, Academic Press, New York, 1989.
[56] 洪青元,應用奈米粒子標記與銀析出偵測法於免疫分析之研究,國立成功大學碩士論文,民國九十四年。
[57] Y. C. Lin, M. Li, C. Y. Wu, W. C. Hsiao and Y. C. Chung, “Microchips for cell-type identification,” The seventh International Symposium on Micro Total Analysis System, μTAS 2003, California, USA, 729, 2003.
[58] http://members.aol.com/logan20/red_tabl.html
[59] 黃浩軒,結合奈米訊號增強技術及阻抗晶片於免疫分析之研究,國立成功大學碩士論文,民國九十五年。
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2012-08-29起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2012-08-29起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw