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研究生: 蕭竹彣
Hsiao, Chu-Wen
論文名稱: 以1-Thio-β-D-glucose修飾螢光金奈米團簇表面於標記腫瘤細胞之應用
Application of 1-Thio-β-D-glucose Modified Fluorescent Gold Nanoclusters for the Tumor Cells Imaging
指導教授: 陳家俊
Chen, Chia-Chun
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2016
畢業學年度: 104
語文別: 中文
論文頁數: 79
中文關鍵詞: 螢光金奈米團簇表面修飾葡萄糖轉運蛋白(GLUT1)1-Thio-β-D-glucose
英文關鍵詞: Fluorescent Gold Nanoclusters, Surface Modification, Glucose Transporter 1 (GLUT1), 1-Thio-β-D-glucose
DOI URL: https://doi.org/10.6345/NTNU202204485
論文種類: 學術論文
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  • 螢光金奈米團簇(Gold Nanoclusters, Au NCs)因其良好的生物
    相容性、易於進行表面修飾及其獨特的光學性質,被廣泛應用於生
    物感測及生物成像。葡萄糖轉運蛋白1(GLUT1)為一種跨膜蛋白,負責運送葡萄糖在血管與細胞之進出。瓦式效應(Warburg effect)指腫瘤細胞即使在有氧的環境下也偏好透過糖解作用勝於氧化磷酸化來產生能量;在腫瘤細胞上,GLUT1會因為瓦式效應而在細胞膜上有大量表現。本實驗以螢光金奈米團簇標記腦瘤細胞,先以還原型的穀胱甘肽與四氯金酸合成出橘紅色的螢光金奈米團簇(OGS-Au NCs),再將1-Thio-β-D-glucose 修飾於螢光金奈米團簇表面,合成出表面接有葡萄糖之螢光金奈米團簇(TGlu-Au NCs),利用腫瘤細胞之瓦式效應,將接有葡萄糖之螢光金奈米團簇通過GLUT1送入腦瘤細胞,以達成標記腦瘤細胞之目的。在共軛焦顯微鏡所觀測到合成之TGlu-Au NCs能有效被人類腦神經膠質瘤細胞U-87 MG吸收。

    Fluorescent gold nanoclusters have been widely employed in the biosensing and bioimaging because of their high biocompatibility, facile surface modification and unique optical properties. Glucose transporter 1 (GLUT-1) is a transmembrane protein which transport of glucose through cell membrane. Warburg Effect describes that tumor cells prefer to glycolysis rather than oxidative phosphorylation for energy production even in the presence of ample oxygen, tumor cells are increased in glucose uptake and elevated glycolysis. In this study, we prepared fluorescent gold nanoclusters and then the surface of fluorescent gold nanoclusters were modified with 1-Thio-β-D-glucose (TGlu-Au NCs). The TGlu-Au NCs showed very low cytotoxicity (the cell viability
    > 90%). The confocal images show that the brain tumor cells of U-87 MG cells could uptake TGlu-Au NCs by GLUT1. To sum up, TGlu-Au NCs could be used as a potential fluorescent probe in the brain tumor cells.

    總目錄 摘要......I Abstract......II 總目錄......III 圖目錄......VI 表目錄......IX 第一章 緒論......1 1-1 前言......1 1-2 奈米材料的特性......3 1-2-1 量子侷限效應 (Quantum Confinement Effect)......3 1-2-2 小尺寸效應 (Small Size Effect)......5 1-2-3 表面效應 (Surface Effects)......7 1-3 金屬奈米粒子......8 1-3-1 金屬奈米粒子之製備......8 1-3-2 金屬奈米粒子之局域表面電漿共振效應......12 第二章 文獻回顧與研究動機......14 2-1 金奈米粒子......14 2-2 金奈米團簇 (Gold Nanoclusters, Au NCs)......16 2-2-1 金奈米團簇之製備......18 2-2-2 單層保護金奈米團簇 (Monolayer-Protected Gold Clusters, Au-MPCs)......19 2-2-3 金奈米團簇之配體置換......23 2-3 螢光金奈米團簇......25 2-3-1 不同模板合成之螢光金奈米團簇......28 2-3-2 螢光金奈米團簇之應用......31 2-4 瓦氏效應 (Warburg Effect)......33 2-5 葡萄糖轉運蛋白1 (GLUT1)......35 第三章 實驗設備與步驟......36 3-1 研究動機......36 3-2 實驗藥品......37 3-3 儀器設備基本原理......38 3-3-1 高速離心機 High Speed Centrifuge......38 3-3-2 紫外光/可見光光譜儀 UV-VIS-NIR Spectrometer......39 3-3-3 光致螢光光譜儀 Photoluminescence Spectrometer, PL ......40 3-3-4 穿透式電子顯微鏡 Transmission Electron Microscopy, TEM......41 3-3-5 掃描式電子顯微鏡 Scanning Electron Microscope, SEM ......42 3-3-6 能量散佈光譜儀 Energy Dispersive Spectrometer, EDS ......42 3-3-7 傅立葉轉換紅外線光譜儀 Fourier Transform Infrared Spectrometer, FT-IR......43 3-3-8 核磁共振光譜儀 Nuclear Magnetic Resonance Spectroscopy, NMR......44 3-3-9 熱重量分析儀 Thermogravimetric Analyzer, TGA......45 3-3-10 共軛焦顯微鏡 Confocal Fluorescence Microscopy......45 3-4 實驗流程......46 3-4-1 合成OGS-Au NCs......46 3-4-2 合成TGlu-Au NCs......47 3-4-3 OGS-Au NCs & TGlu-Au NCs之細胞存活率分析 (MTT assay)......50 3-4-4 OGS-Au NCs & TGlu-Au NCs 細胞標定......52 第四章 結果與討論......53 4-1 OGS-Au NCs之合成......53 4-2 TGlu-Au NCs之合成......55 4-3 傅立葉轉換紅外線光譜......59 4-4 1H-NMR圖譜......60 4-5 TGlu-Au NCs之元素分析鑑定......63 4-6 熱重量分析......64 4-7 TGlu-Au NCs之細胞存活率分析......65 4-8 SVG p12 & U-87 MG細胞標定......67 第五章 結論及未來展望......69 參考文獻......70 附錄......74 圖目錄 圖1-1 粒徑大小不同之膠態金(colloidal gold)呈現不同顏色......2 圖1-2 The Lycurgus Cup (a)反射(Reflected)和(b)透射 (Transmitted)光線......2 圖1-3 奈米材料不同維度之(a)能態密度 Density of Energy States (ρ(E),DOS)和(b)形狀......4 圖1-4 侷域表面電漿 (Localized Surface Plasmon) 示意 圖......13 圖2-1 Turkevich- Frens法所合成之金奈米粒子TEM圖 [四氯金酸(50ml,0.01%wt)加入不同量的檸檬酸鈉(1%wt)] (a)16nm (b)24.5nm (c)41nm (d)71.5nm (e)97.5nm (f)147nm......15 圖2-2 奈米團簇......17 圖2-3 奈米團簇能階離散圖......17 圖2-4 奈米團簇之多種製備方式......18 圖2-5 金奈米團簇之功能化......19 圖2-6 單層保護金奈米團簇(Monolayer-Protected Gold Clusters, Au-MPCs)之發展方向......20 圖2-7 Brust−Schiffrin法示意圖......22 圖2-8 Brust-Schiffrin法合成金奈米團簇之TEM......22 圖2-9 單層保護奈米團簇置換反應......23 圖2-10 以烷基硫醇為保護基之金奈米團簇置換反應示意圖......24 圖2-11 不同配體(ligands)作為保護劑之金奈米團簇......26 圖2-12 螢光金奈米團簇以蝕刻法(Top down)和還原法(Bottom up) 及接上不同保護基之放射光譜圖......27 圖2-13 接上不同保護基之螢光金奈米團簇放射光譜(Emission Wavelength)......27 圖2-14 PAMAM樹枝狀高分子作為模板合成之螢光金奈米團簇 及其激發/放射光譜......28 圖2-15 (a)BSA之(1)粉末(2)水溶液和BSA-Au NCs(3)粉末(4)水溶液 (b) BSA水溶液(藍)和BSA-Au NCs(紅)水溶液之吸收光譜 (虛線)和放射光譜(實線),小圖為BSA-Au NCs之激發光 譜,激發波長470 nm......29 圖2-16 (a)OGS-Au NPs&(b)YGS-Au NPs之激發(綠)/放射(紅)光譜 ......30 圖2-17 OGS-Au NPs&YGS-Au NPs之吸收光譜......30 圖2-18 (a)乳腺上皮細胞(MCF10A)和(b)乳癌細胞(MCF7) 之共軛焦顯微成像......31 圖2-19 CL1-5 cells與(a)AS1411-DA Au NPs和(b)DA-Au NPs 培養進行標定之共軛焦顯微鏡圖(c)小鼠活體螢光測試 ......32 圖2-20 三磷酸腺苷 (Adenosine Triphosphate, ATP)......34 圖2-21 Warburg effect示意圖......34 圖2-22 Glut1 and the Blood-Brain-Barrier (BBB)示意圖 ......35 圖3-1 高速離心機......38 圖3-2 紫外光/可見光/近紅外光光譜儀......39 圖3-3 光致螢光光譜儀......40 圖3-4 穿透式電子顯微鏡......41 圖3-5 傅立葉轉換紅外線光譜儀......43 圖3-6 合成OGS-Au NCs......48 圖3-7 合成TGlu-Au NCs......48 圖3-8 TGlu-Au NCs合成示意圖......49 圖4-1 反應完成之OGS-Au NCs......53 圖4-2 不同倍率之OGS-Au NCs TEM圖......54 圖4-3 OGS-Au NCs之UV光譜圖及PL光譜圖......54 圖4-4 五種不濃度之1-Thio-β-D-glucose與OGS-Au NCs反應24小 時後(濃度由左至右依序為64.12mM、32.06mM、6.412mM 、3.206mM、0.6412mM)......56 圖4-5 五種不同濃度的1-Thio-β-D-glucose 反應合成TGlu-Au NCs 之UV光譜圖......57 圖4-6 五種不同濃度的1-Thio-β-D-glucose 反應合成TGlu-Au NCs 之PL光譜圖......57 圖4-7 不同倍率之TGlu-Au NCs TEM圖......58 圖4-8 OGS-Au NCs & TGlu Au NCs之UV光譜圖及PL光譜圖......58 圖4-9 OGS-Au NCs & TGlu-Au NCs之IR光譜圖......59 圖4-10 L-Glutathione reduced (GSH)之1H-NMR圖譜......60 圖4-11 OGS-Au NCs之1H-NMR圖譜......61 圖4-12 1-Thio-β-D-glucose sodium salt之1H-NMR圖譜......62 圖4-13 TGlu-Au NCs之1H-NMR圖譜......62 圖4-14 TGlu-Au NCs之元素分析圖......63 圖4-15 OGS-Au NCs & TGlu-Au NCs之熱重分析......64 圖4-16 細胞代謝產生Formazan之反應圖......65 圖4-17 不同濃度之OGS-Au NCs & TGlu-Au NCs之細胞存活率 ......66 圖4-18 OGS-Au NCs&TGlu-Au NCs標記SVG p12之共軛焦顯微 成像 (scale bar : 50μm)......67 圖4-19 OGS-Au NCs&TGlu-Au NCs標記U-87 MG之共軛焦顯微 成像 (scale bar : 50μm)......68 表目錄 表3-1 實驗藥品......37 表4-1 TGlu-Au NCs之元素組成百分比......63

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