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研究生： 陳奕穎
Yi-Ying Chen
論文名稱： 螢光奈米鑽石及近紅外光螢光染料之螢光能量共振轉移
Fluorescence resonance energy transfer between fluorescent nanodiamonds and near-infrared dyes
指導教授： 張煥正
Chang, Huan-Cheng
學位類別： 碩士
Master
系所名稱： 化學系
Department of Chemistry
論文出版年： 2010
畢業學年度： 98
語文別： 中文
論文頁數： 94
中文關鍵詞： 奈米鑽石 、螢光能量共振轉移 、螢光顯微鏡 、光漂白
英文關鍵詞： diamond nanoparticle, fluorescence resonance energy transfer, fluoresce microscopy, photobleaching
論文種類： 學術論文
相關次數： 點閱：181  下載：10 
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    • 具有NV0及NV-缺陷中心（defect center）的螢光奈米鑽石（fluorescent nanodiamond），是一種擁有許多獨特特性的新穎奈米材料，例如絕佳的生物相容性、容易進行表面修飾、具有較高組織穿透力的紅色螢光以及優異的光穩定性，很適合用來做為近紅外光螢光生物標記（biolabel）。近來，螢光能量共振轉移廣泛地被應用在研究生物分子結構、酵素動力學及蛋白質交互作用上，因此利用螢光奈米鑽石傑出的光穩定性，做為能量轉移之施子（donor），可提供一個以螢光奈米鑽石為主的生物螢光感測器應用。在本篇論文中，我們藉由測量19奈米直徑螢光奈米鑽石與近紅外光染料（IRDye 800 CW）在不同比例下螢光光譜，以及觀察在聚賴胺酸（poly-lysine）基質（matrix）上，23奈米直徑螢光奈米鑽石之螢光強度及螢光生命期（lifetime）在光漂白近紅外光染料前後之變化，可發現螢光能量轉移效率可達30％。同時，藉由蒙地卡羅模擬（Monte Carlo simulation）可估算平均每個螢光奈米鑽石微粒可與鄰近10個染料分子發生螢光能量共振轉移。這對於利用螢光奈米鑽石做為單分子螢光標記或具有奈米級解析度之分子尺（molecular ruler）提供了新的應用平台。

    Fluorescent nanodiamond (FND) is a potent fluorescent probe possessing several unique properties such as　excellent biocompatibility, facile surface modification, high tissue-penetrable red fluorescence, and outstanding photostability. The neutral and negatively charged nitrogen-vacancy (NV0 and NV-) defect centers embedded in the　diamond lattice is responsible for the far red photon emission from this novel nanomaterial. FNDs are excellent biolabels and could be used to probe intricate biochemical processes. Fluorescence resonance energy transfer (FRET) has recently been widely introduced to study biomolecular configuration, enzyme kinetics and protein-protein interactions. Therefore, as a donor, with its perfect photostability, FND paves the way for a FND-based biosensor. In this thesis, we investigate the fluorescence spectra of 19-nm-sized FND conjugated with near-infrared dye (IRDye 800 CW) and demonstrate that it is possible to approach the FRET efficiency up to 30% between 23-nm-sized FND and dye molecules co-embedded in a poly-lysine matrix. We measured the changes in both fluorescence intensity and lifetime of FNDs before and after photobleaching of near-infrared dye. Moreover, according to Monte Carlo simulations, on an average, each FND transfers its energy to 10 dye molecules attached to its periphery. These results set the stage for FND-based single particle bio-labeling or molecular ruler with nanometric resolution.

    目 錄 ABSTRACT I 摘 要 II ACKNOWLEDGEMENT III 致 謝 VI 目 錄 VIII 圖 目 錄 XII 表 目 錄 XV 第一章 緒論 - 1 - 第二章 文獻回顧 - 5 - 2.1 螢光奈米鑽石（FLUORESCENT NANODIAMOND） - 5 - 2.1.1 結構特性 - 5 - 2.1.2 種類 - 7 - 2.1.3 缺陷中心（defect center）24, 25 - 8 - 2.1.4 應用性質 - 10 - 2.2 螢光能量共振轉移（FLUORESCENCE RESONANCE ENERGY TRANSFER） - 12 - 2.3 研究動機 - 20 - 第三章 實驗藥品與儀器介紹 - 22 - 3.1 實驗藥品 - 22 - 3.2 實驗儀器 - 23 - 3.2.1 管狀高溫爐（Tube furnace）： Thermo Scientific Thermolyne TL-21130-33 - 23 - 3.2.2 離子束佈植裝置（Ion beam implantation）42 - 24 - 3.2.3 聚焦微波化學反應系統（Focused MicrowaveTM Synthesis System）：CEM Discover discover 1 - 27 - 3.2.4 真空迴旋濃縮儀（Rotary evaporator）： Panchum Scientific Corp. 2781070 - 28 - 3.2.5 紫外光可見光光譜儀（UV-Vis spectrophotometer）：Hitachi U-3310 - 29 - 3.2.6 微量冷凍離心機（Refrigerated centrifuge）： Kubota 3750 - 31 - 3.2.7 微量超高速離心機（Micro ultracentrifuge）： Hitachi CS120GXL - 33 - 3.2.8 超音波震盪器（Sonicator）： Delta DC150 - 33 - 3.2.9 試管震盪器（Shaker）： Scientific industries Vortex-Genie 2 - 33 - 3.2.10 二次水製造機： Milipore Simplicity® - 33 - 3.2.11 加熱攪拌器（Stirrer/hot plate）： Corning PC-420 - 33 - 3.2.12 動態光散射粒徑分析儀（Delsa™Nano Zeta Potential and Submicron Particle Size Analyzer） - 34 - 3.2.13 光譜儀（Multichannel spectrometer）： Hamamatsu C7374 - 38 - 3.2.14 螢光顯微鏡（Fluorescent microscopy）： Nikon E600 - 39 - 3.2.15 時間相關單光子計數器（Time Correlated Single Photon Counting）：PicoHarp 300 - 41 - 第四章 實驗方法 - 44 - 4.1 螢光奈米鑽石樣品製備 - 44 - 4.1.1 五十奈米螢光奈米鑽石製備 - 44 - 4.1.2 三十奈米螢光奈米鑽石製備 - 46 - 4.2 差速離心法（DIFFERENTIAL CENTRIFUGATION）分離不同粒徑之螢光奈米鑽石 - 47 - 4.3 螢光奈米鑽石表面修飾與染料耦合(CONJUGATION) - 49 - 4.3.1 以乙二胺（ethylenediamine）作為鏈接分子（linker） - 49 - 4.3.2 以半胱胺（cysteamine）作為鏈接分子 - 50 - 4.3.3 將染料與螢光奈米鑽石共嵌（co-embedded）於聚賴胺酸（poly-lysine）基質中 - 51 - 4.4 螢光光譜測量 - 53 - 4.5 藉由時間相關單光子計數及受子光漂白（ACCEPTOR PHOTOBLEACHING）測量螢光生命期（FLUORESCENCE LIFETIME） - 55 - 4.6 蒙地卡羅模擬（MONTE CARLO SIMULATION）58, 59 - 59 - 第五章 結果與討論 - 64 - 5.1 螢光奈米鑽石的物理及光學性質 - 64 - 5.1.1 螢光奈米鑽石的螢光光譜 - 64 - 5.1.2 由差速離心法分離後螢光奈米鑽石的粒徑分佈及螢光光譜 - 66 - 5.2 近紅外螢光染料的光學性質 - 72 - 5.3 螢光能量共振轉移光譜 - 73 - 5.3.1 螢光能量共振轉移光譜與計算 - 73 - 5.3.2 螢光能量共振轉移效率 - 76 - 5.4 螢光奈米鑽石生命期測量 - 79 - 5.4.1 螢光奈米鑽石在受子光漂白前後之螢光生命期變化 - 79 - 5.4.2 螢光奈米鑽石之螢光能量共振轉移效率分析 - 81 - 5.5 蒙地卡羅模擬 - 83 - 第六章 結論 - 86 - 第七章 參考文獻 - 88 -   圖 目 錄 圖 1 帶負電的氮原子—空缺螢光缺陷中心之吸收與放光光譜 - 3 - 圖 2 鑽石與石墨結構圖 - 6 - 圖 3 鑽石中各式含氮缺陷圖 - 9 - 圖 4 吸收、螢光與磷光圖 - 13 - 圖 5 螢光能量共振轉移動力學關係圖 - 14 - 圖 6 施子與受子量子能階圖 - 15 - 圖 7 福斯特半徑示意圖 - 18 - 圖 8 管狀高溫爐 - 23 - 圖 9 具有質量選擇器的離子佈植裝置示意圖 - 24 - 圖 10 離子佈植裝置全貌 - 25 - 圖 11 離子束佈植裝置示意圖 - 26 - 圖 12 CEM DISCOVER 1 裝置圖 - 27 - 圖 13 微波加熱示意圖 - 27 - 圖 14 真空迴旋濃縮儀示意圖 - 28 - 圖 15 紫外光可見光光譜儀 （HITACHI U-3310） - 29 - 圖 16 微量冷凍離心機 - 31 - 圖 17 離心過程粒子受力圖 - 32 - 圖 18 分離時間關係式 - 32 - 圖 19 動態光散射與自相關函數圖 - 35 - 圖 20 螢光光譜儀光路設計圖 - 38 - 圖 21 螢光顯微鏡光路圖 - 39 - 圖 22 時間相關單光子計數法概念圖 - 41 - 圖 23 時間相關單光子計數器將訊號轉換成螢光生命期圖 - 42 - 圖 24 螢光奈米鑽石製備流程示意圖 - 45 - 圖 25 差速離心法分離不同粒徑鑽石示意圖 - 48 - 圖 26 螢光奈米鑽石與螢光染料反應流程（乙二胺鏈接）示意圖 - 50 - 圖 27 螢光奈米鑽石與螢光染料反應流程（半胱胺鏈接）示意圖 - 51 - 圖 28 染料與螢光奈米鑽石共嵌流程示意圖 - 52 - 圖 29 染料與螢光鑽石共嵌於基質示意圖 - 52 - 圖 30 螢光光譜儀全貌 - 54 - 圖 31 時間相關單光子計數裝置圖 - 56 - 圖 32 螢光奈米鑽石濾片測試光譜 - 57 - 圖 33 近紅外光螢光染料光漂白測試光譜 - 58 - 圖 34 蒙地卡羅模擬螢光共振能量轉移流程圖 - 59 - 圖 35 螢光奈米鑽石懸浮水溶液之螢光光譜圖。 - 64 - 圖 36 在室溫與低溫下單一（氮—空缺）螢光缺陷中心的螢光光譜 - 65 - 圖 37 不同粒徑螢光奈米鑽石之螢光光譜 - 66 - 圖 38 五十奈米鑽石粒徑分布圖 - 68 - 圖 39 二十一奈米螢光奈米鑽石粒徑分布圖 - 69 - 圖 40 七奈米螢光奈米鑽石粒徑分布圖 - 70 - 圖 41 二十三奈米螢光奈米鑽石粒徑分布圖 - 71 - 圖 42 螢光奈米鑽石與染料吸收及螢光光譜重疊圖 - 72 - 圖 43 十九奈米螢光奈米鑽石與染料的螢光能量共振轉移光譜 - 74 - 圖 44 七奈米螢光奈米鑽石與染料的螢光能量共振轉移光譜 - 75 - 圖 45 不同比例之十九奈米螢光奈米鑽石的螢光能量共振轉移效率 - 76 - 圖 46 不同比例之七奈米螢光奈米鑽石的螢光能量共振轉移效率 - 77 - 圖 47 螢光奈米鑽石在光漂白前後的螢光光譜圖 - 80 - 圖 48 螢光奈米鑽石在光漂白前後的螢光生命期圖 - 81 - 圖 49 螢光能量共振轉移效率統計分析圖 - 82 - 圖 50 螢光能量共振轉移效率對奈米鑽石半徑之蒙地卡羅模擬圖 - 83 - 表 目 錄 表 1 鑽石種類表 - 7 - 表 2 鑽石的缺陷中心性質表 - 9 - 表 3 奈米鑽石的應用 - 11 - 表 4 高解析單分子測量技術比較表 - 19 -

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