簡易檢索 / 詳目顯示

回結果列表
研究生： 蔣馥如
Chiang, Fu-Ju
論文名稱： 萬古黴素-磁性氧化石墨烯作為快速檢測與鑑定細菌之生物感測器
Vancomycin-Magnetic Graphene Oxide Based Biosensor for Rapid Detection and Identification of Bacteria
指導教授： 凌永健
Ling, Yong-Chien
口試委員: 劉銘龍
陳貴通
凌永健
學位類別： 碩士
Master
系所名稱： 理學院 - 化學系
Department of Chemistry
論文出版年： 2013
畢業學年度： 101
語文別： 中文
論文頁數： 92
中文關鍵詞： 石墨烯 、萬古黴素 、螢光共振能量轉移 、基質輔助雷射脫附游離-飛行時間式質譜法 、細菌
外文關鍵詞： graphene, vancomycin, fluorescence resonance energy transfer, matrix assisted laser desorption ionization-time of flight mass spectrometry, bacteria
相關次數： 點閱：3  下載：0 
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報

    • 傳統鑑定細菌之方法大多需要數小時至數天的培養時間，過程繁複且耗時，臨床診斷上發展快速且靈敏的細菌檢測法成為目前時代潮流的趨勢。
    本研究成功製備萬古黴素-磁性氧化石墨烯(Vancomycin- Magnetic Graphene Oxide，MGOVan)與二氧化矽奈米粒子(Silica Nanoparticles，SiNPs)作為快速偵測細菌之生物感測器。萬古黴素有助於溶液中有效抓取目標菌種。利用Stöber方法製備的SiNPs鍛燒後具有螢光特性。SiNPs與MGOVan相靠近時，產生螢光共振能量轉移(Fluorescence Resonance Energy Transfer，FRET)之作用，因此，藉由螢光強度之改變可偵測樣品中細菌濃度，對金黃色葡萄球菌與大腸桿菌之偵測極限約為20 cfu/mL。
    利用MGO的超順磁之特性，在外加磁場的輔助下可將細菌快速從溶液中分離出來，透 過基質輔助雷射脫附游離-飛行時間式質譜儀(Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometer，MALDI-TOFMS)的分析，可以鑑定細菌種類，對金黃色葡萄球菌與大腸桿菌之偵測極限約為108 cfu/mL。本研究亦製備萬古黴素與芥子酸-磁性氧化石墨烯(Vancomycin and Sinapinic Acid-Magnetic Graphene Oxide，MGOVanSA)，增加樣品製備中基質的比例，製備三明治結構樣品，提升基質傳遞雷射能量給細菌的效率，有助於細菌脫附離子化。本研究利用螢光改變與質譜鑑定細菌菌種，使用螢光低偵測極限作為初篩工具，輔以高解析能力質譜作為確認工具，深具應用於食物與水樣中的病原菌偵測之潛力。

    Conventional analytical techniques employed for bacteria detection are time consuming (several hours to days). There is an increasing need for the development of rapid and sensitive methods for bacterial detection in clinical diagnosis.
    In this work, efforts were taken to synthesize graphene based biosensor vancomycin-magnetic graphene oxide (MGOVan) with silica nanoparticles (SiNPs) as biosensors for rapid detection of bacteria. Vancomycin has the capacity of capturing bacteria from aqueous solution. SiNPs were prepared by the Stöber method and then calcining which led to fluorescent. Using fluorescence resonance energy transfer (FRET) from SiNPs to MGOVan, the change in fluorescent intensity offered a novel method for bacteria detection. The lowest detectable bacteria concentration for both S. aureus and E. coli in aqueous solution was about 20 cfu/mL.
    Further, with unique magnetic feature, MGOVan-bacteria conjugates were aggregated under the external magnet quickly and subjected to matrix assisted laser desorption ionization-time of flight mass spectrometer (MALDI-TOFMS) to distinguish bacteria species. The detectable bacteria concentration for both S. aureus and E. coli by MALDI-TOFMS was about 108 cfu/mL. We also prepared vancomycin and sinapinic acid-magnetic graphene oxide (MGOVanSA). MGOVanSA can enhance bacteria desorption and ionization efficiencies by larger SA ratio in MALDI sample and sample’s sandwich composition. The change in fluorescent intensity and mass spectrometry results demonstrated that MGOVan is an ideal candidate for rapid detection of food and waterborn pathogens.

    第一章 緒論 1 1.1 研究動機與目的 1 1.2 食品中毒簡介 2 1.3 食品微生物檢驗方法 4 1.3.1 傳統細菌檢驗方法 4 1.3.2 其他快速細菌檢驗方法 6 1.3.3 質譜技術於生化應用的發展 10 1.4 石墨烯 12 1.5 二氧化矽奈米粒子 13 1.5.1 二氧化矽奈米粒子簡介 13 1.5.2 二氧化矽奈米粒子之光學性質 14 1.6 磁性奈米粒子 15 第二章 螢光共振能量轉移 16 2.1 螢光與磷光原理 16 2.2 螢光共振能量轉移 17 2.3 細菌發光感測器之螢光共振能量轉移 18 第三章 基質輔助雷射脫附游離－飛行時間式質譜法之介紹 19 3.1 MALDI-TOFMS 發展歷史 19 3.2 MALDI-TOFMS樣品製備與基質特性 20 3.2.1 MALDI-TOFMS樣品製備 20 3.2.2 MALDI-TOFMS基質特性 20 3.3 MALDI形成離子機制 21 3.4 MALDI-TOFMS儀器原理 22 第四章 實驗方法 24 4.1 MGOVan / MGOVanSA細菌探針之製備 24 4.1.1 氧化石墨烯之製備 25 4.1.2 磁性氧化石墨烯之製備 25 4.1.3 磁性氧化石墨烯之修飾 25 4.2 MGOVan / MGOVanSA細菌探針特性分析 28 4.3 發光二氧化矽奈米粒子之製備 30 4.3.1 二氧化矽奈米粒子之製備 30 4.3.2 發光二氧化矽奈米粒子之製備 31 4.4 發光二氧化矽奈米粒子之特性分析 31 4.5 細菌樣品分析 32 4.5.1 藥品配製 33 4.5.2 細菌樣品 34 4.5.3 菌落數試驗 34 4.6 MGOVan細菌探針抓取細菌 35 4.6.1 MGOVan細菌探針抓取細菌方法 36 4.6.2 MGOVan細菌探針抓取細菌效能 36 4.6.3 以SEM證明MGOVan細菌探針抓取細菌效果 37 4.7 細菌發光感測器分析細菌樣品 37 4.7.1 細菌發光感測器之製備 38 4.7.2 細菌發光感測器分析細菌樣品 38 4.8 MALDI-TOFMS鑑定樣品細菌種類 39 4.8.1 基質溶液配製 39 4.8.2 MALDI-TOFMS直接分析菌種及其偵測極限 39 4.8.3 MALDI-TOFMS確認細菌探針抓取細菌之菌種與偵測極限 40 4.8.4 MALDI-TOFMS操作條件 41 4.8.5 蛋白質資料庫搜尋 41 第五章 結果與討論 42 5.1 修飾萬古黴素於磁性氧化石墨烯修飾量之探討 42 5.2 MGOVan/MGOVanSA細菌探針之特性分析 44 5.3 二氧化矽奈米粒子之特性分析 51 5.4 細菌樣品分析－菌落數試驗 56 5.5 MGOVan細菌探針抓取細菌 61 5.5.1 MGO與MGOVan抓取細菌能力之探討 62 5.5.2 MGOVan細菌探針抓取細菌之最佳化 64 5.5.3 以SEM確認MGOVan抓取細菌的效果 68 5.6 細菌發光感測器之細菌樣品分析 69 5.7 MALDI-TOFMS鑑定樣品細菌種類之探討 75 5.7.1 以MALDI-TOFMS直接分析菌種 75 5.7.2 MGOVan細菌探針抓取濃縮細菌進行MALDI-TOFMS分析 78 5.7.3 MGOVan/ MGOVanSA抓取濃縮金黃色葡萄球菌以MALDI-TOFMS分析 82 第六章 結論 85 第七章 參考文獻 87

    1. Gu, H. W.; Ho, P. L.; Tsang, K. W. T.; Wang, L.; Xu, B., Using biofunctional magnetic nanoparticles to capture vancomycin-resistant enterococci and other gram-positive bacteria at ultralow concentration. Journal of the American Chemical Society 2003, 125 (51), 15702-15703.
    2. 行政院衛生署 http://www.doh.gov.tw/. (上網日期：2013/08/25)
    3. Mims, C. P., J.; Roitt,I.; Wakelin, D.; Williams R., Medical microbiology, C.V. Mosby: 1998.
    4. 劉向寧, 吳靖宙, 張長泉,張憲彰, 生物晶片與食品微生物檢測. 國際農業科技新知 2003, 17.
    5. 中華民國國家標準法(CNS)網路檢索系統 http://www.cnsonline.com.tw/. (上網日期：2013/08/25)
    6. Perry, J. D.; Freydiere, A. M., The application of chromogenic media in clinical microbiology. Journal of Applied Microbiology 2007, 103 (6), 2046-2055.
    7. Sapsford, K. E.; Bradburne, C.; Delehanty, J. B.; Medintz, I. L., Sensors for detecting biological agents. Materials Today 2008, 11 (3), 38-49.
    8. 張雯惠，林俊宏, 聚合酶連鎖反應簡介. 奈米通訊 2005, 12 (1), 6-11.
    9. Fricker, M.; Messelhausser, U.; Busch, U.; Scherer, S.; Ehling-Schulz, M., Diagnostic real-time PCR assays for the detection of emetic Bacillus cereus strains in foods and recent food-borne outbreaks. Applied and Environmental Microbiology 2007, 73 (6), 1892-1898.
    10. Huletsky, A.; Giroux, R.; Rossbach, V.; Gagnon, M.; Vaillancourt, M.; Bernier, M.; Gagnon, F.; Truchon, K.; Bastien, M.; Picard, F. J.; van Belkum, A.; Ouellette, M.; Roy, P. H.; Bergeron, M. G., New real-time PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus directly from specimens containing a mixture of staphylococci. Journal of Clinical Microbiology 2004, 42 (5), 1875-1884.
    11. Liu, R. H.; Yang, J. N.; Lenigk, R.; Bonanno, J.; Grodzinski, P., Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection. Analytical Chemistry 2004, 76 (7), 1824-1831.
    12. 劉定宇; 王懷賢; 蔡焜棟; 陳玉; 陳友暄; 趙元駿; 張軒豪; 韓吟宜; 連偉男; 林奇宏; 王俊凱; 王玉麟, 奈米光譜技術平台之快速微生物檢測晶片. 臺灣醫學 2012, 16 (3), 271-283.
    13. Anhalt, J. P.; Fenselau, C., IDENTIFICATION OF BACTERIA USING MASS-SPECTROMETRY. Analytical Chemistry 1975, 47 (2), 219-225.
    14. Fox, A.; Black, G. E.; Fox, K.; Rostovtseva, S., DETERMINATION OF CARBOHYDRATE PROFILES OF BACILLUS-ANTHRACIS AND BACILLUS-CEREUS INCLUDING IDENTIFICATION OF O-METHYL METHYLPENTOSES BY USING GAS-CHROMATOGRAPHY MASS-SPECTROMETRY. Journal of Clinical Microbiology 1993, 31 (4), 887-894.
    15. Heller, D. N.; Murphy, C. M.; Cotter, R. J.; Fenselau, C.; Uy, O. M., Constant neutral loss scanning for the characterization of bacterial phospholipids desorbed by fast atom bombardment. Analytical Chemistry 1988, 60 (24), 2787-2791.
    16. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M., ELECTROSPRAY IONIZATION FOR MASS-SPECTROMETRY OF LARGE BIOMOLECULES. Science 1989, 246 (4926), 64-71.
    17. Bakhtiar, R.; Nelson, R. W., Mass spectrometry of the proteome. Molecular Pharmacology 2001, 60 (3), 405-415.
    18. Ho, Y. P.; Hsu, P. H., Investigating the effects of protein patterns on microorganism identification by high-performance liquid chromatography-mass spectrometry and protein database searches. Journal of Chromatography A 2002, 976 (1-2), 103-111.
    19. Geim, A. K.; Novoselov, K. S., The rise of graphene. Nature Materials 2007, 6 (3), 183-191.
    20. Lee, C.; Wei, X. D.; Kysar, J. W.; Hone, J., Measurement of the elastic properties and intrinsic strength of monolayer graphene. Science 2008, 321 (5887), 385-388.
    21. Chen, J. H.; Jang, C.; Xiao, S. D.; Ishigami, M.; Fuhrer, M. S., Intrinsic and extrinsic performance limits of graphene devices on SiO2. Nature Nanotechnology 2008, 3 (4), 206-209.
    22. Schedin, F.; Geim, A. K.; Morozov, S. V.; Hill, E. W.; Blake, P.; Katsnelson, M. I.; Novoselov, K. S., Detection of individual gas molecules adsorbed on graphene. Nature Materials 2007, 6 (9), 652-655.
    23. (a) Kang, X. H.; Wang, J.; Wu, H.; Aksay, I. A.; Liu, J.; Lin, Y. H., Glucose Oxidase-graphene-chitosan modified electrode for direct electrochemistry and glucose sensing. Biosensors & Bioelectronics 2009, 25 (4), 901-905; (b) Shan, C. S.; Yang, H. F.; Song, J. F.; Han, D. X.; Ivaska, A.; Niu, L., Direct Electrochemistry of Glucose Oxidase and Biosensing for Glucose Based on Graphene. Analytical Chemistry 2009, 81 (6), 2378-2382.
    24. Haixin Chang, L. T., Ying Wang, Jianhui Jiang, and Jinghong Li, Graphene Fluorescence Resonance Energy Transfer Aptasensor for the Thrombin Detection. Analytical Chemistry 2010, 82 (6), 2341-2346.
    25. W. Stöber, A. F., E. Bohn, Controlled Growth of Monodisperse Silica Spheres in the Micron Size Range. JOURNAL OF COLLOID AND INTERFACE SCIENCE 1968, 26, 62-69.
    26. Vallet-Regi, M.; Balas, F.; Arcos, D., Mesoporous materials for drug delivery. Angewandte Chemie-International Edition 2007, 46 (40), 7548-7558.
    27. Rossi, L. M.; Shi, L.; Quina, F. H.; Rosenzweig, Z., Stöber Synthesis of Monodispersed Luminescent Silica Nanoparticles for Bioanalytical Assays. Langmuir 2005, 21 (10), 4277-4280.
    28. Van Blaaderen, A.; Vrij, A., Synthesis and characterization of colloidal dispersions of fluorescent, monodisperse silica spheres. Langmuir 1992, 8 (12), 2921-2931.
    29. Enrichi, F.; Trave, E.; Bersani, M., Acid synthesis of luminescent amine-functionalized or erbium-doped silica spheres for biological applications. Journal of Fluorescence 2008, 18 (2), 507-511.
    30. Chan, Y.; Zimmer, J. P.; Stroh, M.; Steckel, J. S.; Jain, R. K.; Bawendi, M. G., Incorporation of Luminescent Nanocrystals into Monodisperse Core–Shell Silica Microspheres. Advanced Materials 2004, 16 (23-24), 2092-2097.
    31. Schmedake, A. M. J. a. T. A., A Novel Approach to Monodisperse, Luminescent Silica Spheres. Chem. Mater. 2006, 18, 3173-3175.
    32. 陳建淼, 洪連輝. 磁性物質（Magnetic Materials）. http://case.ntu.edu.tw/hs/wordpress/. (上網日期：2013/08/25)
    33. Kumar, R. V.; Koltypin, Y.; Cohen, Y. S.; Cohen, Y.; Aurbach, D.; Palchik, O.; Felner, I.; Gedanken, A., Preparation of amorphous magnetite nanoparticles embedded in polyvinyl alcohol using ultrasound radiation. Journal of Materials Chemistry 2000, 10 (5), 1125-1129.
    34. Vogel, S. S.; Thaler, C.; Koushik, S. V., Fanciful FRET. Sci. STKE 2006, 331, re2-.
    35. Jaemyung Kim, L. J. C., Franklin Kim, and Jiaxing Huang, Visualizing Graphene Based Sheets by Fluorescence Quenching Microscopy. J. AM. CHEM. SOC. 2010, 132, 260-267.
    36. Posthumus, M. A.; Kistemaker, P. G.; Meuzelaar, H. L. C.; Ten Noever de Brauw, M. C., Laser desorption-mass spectrometry of polar nonvolatile bio-organic molecules. Analytical Chemistry 1978, 50 (7), 985-991.
    37. Karas, M.; Bachmann, D.; Hillenkamp, F., Influence of the wavelength in high-irradiance ultraviolet laser desorption mass spectrometry of organic molecules. Analytical Chemistry 1985, 57 (14), 2935-2939.
    38. Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T.; Matsuo, T., Protein and polymer analyses up to m/z 100 000 by laser ionization time-of-flight mass spectrometry. Rapid Communications in Mass Spectrometry 1988, 2 (8), 151-153.
    39. Karas, M.; Hillenkamp, F., Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Analytical Chemistry 1988, 60 (20), 2299-2301.
    40. Bahr, U.; Karas, M.; Hillenkamp, F., Analysis of biopolymers by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Fresenius' Journal of Analytical Chemistry 1994, 348 (12), 783-791.
    41. Ehring, H.; Karas, M.; Hillenkamp, F., Role of photoionization and photochemistry in ionization processes of organic molecules and relevance for matrix-assisted laser desorption lonization mass spectrometry. Organic Mass Spectrometry 1992, 27 (4), 472-480.
    42. Zenobi, R.; Knochenmuss, R., Ion formation in MALDI mass spectrometry. Mass Spectrometry Reviews 1998, 17 (5), 337-366.
    43. Hummers, W. S.; Offeman, R. E., Preparation of Graphitic Oxide. Journal of the American Chemical Society 1958, 80 (6), 1339-1339.
    44. Kell, A. J.; Simard, B., Vancomycin architecture dependence on the capture efficiency of antibody-modified microbeads by magnetic nanoparticles. Chemical Communications 2007, (12), 1227-1229.
    45. (a) Tuinstra, F.; Koenig, J. L., RAMAN SPECTRUM OF GRAPHITE. Journal of Chemical Physics 1970, 53 (3), 1126-&; (b) Ferrari, A. C.; Robertson, J., Interpretation of Raman spectra of disordered and amorphous carbon. Physical Review B 2000, 61 (20), 14095-14107.
    46. Green, W. H.; Le, K. P.; Grey, J.; Au, T. T.; Sailor, M. J., White phosphors from a silicate-carboxylate sol-gel precursor that lack metal activator ions. Science 1997, 276 (5320), 1826-1828.
    47. Wang, L.; Estevez, M. C.; O'Donoghue, M.; Tan, W. H., Fluorophore-free luminescent organosilica nanoparticles. Langmuir 2008, 24 (5), 1635-1639.
    48. Akhavan, O.; Ghaderi, E.; Esfandiar, A., Wrapping Bacteria by Graphene Nanosheets for Isolation from Environment, Reactivation by Sonication, and Inactivation by Near-Infrared Irradiation. Journal of Physical Chemistry B 2011, 115 (19), 6279-6288.
    49. Baron, S., Medical Microbiology, 4th edition. 1996.
    50. BACTERIA: Staphylococcus aureus. http://www.engr.psu.edu/iec/abe/database/bStaphA.htm. (上網日期：2013/08/25)
    51. B10NUMB3R5 (The Database of Useful Biological Numbers). www.bioNumbers.org. (上網日期：2013/08/25)

    無法下載圖示 全文公開日期 本全文未授權公開 (校內網路)
    全文公開日期 本全文未授權公開 (校外網路)

    QR CODE