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研究生: 賴盈禎
Lai, Ying-Chen
論文名稱: 以聚脯胺酸調控色胺酸與金奈米粒子之距離探討螢光淬熄效應
Distance-dependent Fluorescence Quenching of Tryptophan by Gold Nanoparticles within 5 nm Spaced by Polyprolines
指導教授: 朱立岡
Chu, Li-Kang
口試委員: 洪嘉呈
Horng, Jia-Cherng
陳仁焜
Chen, Jen-Kun
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: 2017
畢業學年度: 106
語文別: 中文
論文頁數: 124
中文關鍵詞: 聚脯胺酸色胺酸金奈米粒子螢光淬熄
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  • 當金奈米粒子靠近螢光分子時,能誘發其螢光淬熄效應,且此效應與兩者的距離有關,但目前尚無對短距離(< 5 nm)進行系統性的研究。在此,吾人選用色胺酸作為螢光分子,以及利用聚脯胺酸(polyproline,PP)於水溶液中形成堅固的PPII結構作為分子尺(molecular ruler),並藉由調整脯胺酸的數量,以控制金奈米粒子與色胺酸距離在5 nm內,探討金奈米粒子對螢光的淬熄效應。

    吾人合成四種不同長度的胜肽,序列為CAPnWN,其中n=3、6、10、13,並成功地將其透過半胱胺酸的硫醇基,以金硫共價鍵鍵結於金奈米粒子表面。以靜態紫外/可見光吸收光譜、螢光光譜、衰減全反射式紅外吸收光譜以及穿透式電子顯微鏡,觀察其合成前後形貌的變化。除了靜態光譜資訊,吾人亦利用時間相關單光子技術偵測其螢光生命期,以266 nm 的雷射激發,收集355–365 nm的螢光訊號。吾人發現色胺酸在CAPnWN中的螢光生命期較其單體短,而當CAPnWN被修飾於金奈米粒子表面後,其色胺酸的螢光生命期隨著與金奈米粒子的距離越近而越短。若以奈米表面能量轉移機制分析此距離相關的螢光衰減速率,得到能量轉移速率與距離倒數呈現1.3次方關係,此結果與理論值4差異過大,因此吾人認為奈米表面能量轉移非主因。若以電子轉移機制分析,並考慮色胺酸彼此靠近可能有π-π 堆疊的影響,可以衰減常數0.1 Å-1擬合吾人數據,與以往文獻一致。故吾人認為色胺酸螢光淬熄的主因為電子轉移,以聚脯胺酸的胜肽骨架作為跳板,金奈米粒子作為電子受體。故由本實驗得知,聚脯胺酸不僅可作為分子尺,亦可作為電子轉移的模板。


    Gold nanoparticles (AuNPs) often induce the quenching of excited states of fluorophores when they are close. The quenching efficiency has been observed as a function of distance between the AuNP and the fluorophore. However, there has not been discussed about AuNP-induced quenching within 5 nm. In this work, tryptophan is served as the fluorophore to study the AuNP-induced quenching and polyprolines (PP), which form the rigid PPII structure in solution, are used as molecular rulers to separate the AuNP and Trp by tuning the numbers of the proline units within 5 nm.
    The surfaces of the AuNP were successfully anchored with peptides, CAPnWN which n=3, 6, 10, 13, through the terminal cysteine to form the Au-S bonding. The optical properties of AuNP-CAPnWN were characterized by UV/Visible absorption spectroscopy, fluorescence spectroscopy, ATR-FTIR and TEM. Besides the steady-state optical characterization, the temporal profiles of the tryptophan fluorescence at 355-365 nm upon 266 nm excitation were collected with the time-correlated single-photon counting. We found that the fluorescence kinetics of tryptophan in CAPnWN is slightly accelerated in comparison with the pure tryptophan. As the CAPnWN are attached onto the gold nanoparticles, the fluorescence kinetics becomes more accelerated as the separation becomes shorter. Both energy-transfer and electron-transfer processes are considered to be deactivation pathways. However, the energy transfer rate depends on the 1/d1.3 separation distance which is far from the nanosurface energy transfer mechanism. Therefore, the energy transfer from Trp to AuNP is not the major reason. Then, considering the electron transfer rate (k_ET) as an exponential function of the peptide chain length (k_ET∝e^(-βd)), and the π-stacking interaction between adjacent CAPnWN, the β could be fitted as 0.1 Å-1 same as previous studies. As a result, the fluorescence quenching of Trp by AuNP depends on their distance is attributed to the electron transfer mechanism. The polyprolines are served as stepping stones and the AuNPs are the electron acceptors. It is noteworthy that the polyproline can not only be the molecular ruler but also be the relay for electron transfer.

    第一章 緒論 1 1.1前言 1 1.2文獻簡介 1 1.3實驗動機與目的 2 參考文獻 5 第二章 金屬奈米粒子與多胜肽的性質 9 2.1金屬奈米粒子 9 2.1.1金屬奈米粒子的光學性質 9 2.1.2金屬奈米粒子誘發的螢光淬熄機制 10 2.2色胺酸的螢光性質 11 2.2.1色胺酸的螢光機制 11 2.2.2色胺酸於胜肽或蛋白質中螢光性質的變化 12 2.3聚脯胺酸的性質 13 2.3.1聚脯胺酸的二級結構 13 2.3.2聚脯胺酸作為間隔分子運用在福斯特共振能量轉移 14 2.4多胜肽的電子轉移機制 14 2.4.1電子轉移理論 14 2.4.2多胜肽中的電子轉移機制與影響因素 16 2.5固相胜肽合成法 18 2.5.1樹脂與連接體 18 2.5.2去保護 19 2.5.3活化 19 2.5.4耦合 20 2.5.5切除 20 2.6金奈米粒子表面修飾胜肽 20 參考文獻 39 第三章 儀器原理與樣品製備 46 3.1儀器介紹 46 3.1.1靜態紫外/可見光吸收光譜 (UV/Visible absorption spectroscopy,UV/Vis) 46 3.1.2圓二色光譜 (Circular dichroism spectroscopy,CD) 47 3.1.3螢光分光光譜 (Fluorescence spectroscopy,FL) 48 3.1.4衰減全反射式傅氏轉換紅外光譜儀(Attenuated total reflection Fourier transform infrared spectroscopy,ATR-FTIR) 49 3.1.5穿透式電子顯微鏡(Transmission electron microscopy,TEM) 52 3.1.6時間相關單光子計數系統 (Time-correlated single photon counting,TCSPC) 53 3.2樣品合成與製備 54 3.2.1金奈米粒子合成 54 3.2.2胜肽合成 55 3.2.3樹脂切除與胜肽純化 56 3.2.4胜肽純度與特徵光譜鑑定 56 3.2.5金奈米粒子表面修飾胜肽 57 3.3儀器型號與參數設定 58 3.3.1微波胜肽合成儀 58 3.3.2高效能液相層析儀 58 3.3.3基質輔助雷射脫附游離飛行時間質譜儀 58 3.3.4冷凍乾燥機 58 3.3.5可控溫高速離心機和微量離心機 59 3.3.6靜態紫外/可見光吸收光譜 59 3.3.7圓二色光譜 59 3.3.8螢光分光光譜 59 3.3.9衰減全反射式傅氏轉換紅外光譜儀 60 3.3.10穿透式電子顯微鏡 60 3.3.11時間相關單光子計數系統 60 參考文獻 77 第四章 實驗結果與討論 78 4.1胜肽結構鑑定 78 4.1.1 CAPnWN的圓二色光譜 78 4.1.2 CAPnWN的衰減全反射式傅氏轉換紅外吸收光譜 78 4.1.3 CAPnWN的長度估算 79 4.2金奈米粒子表面修飾CAPnWN之靜態光譜鑑定 80 4.2.1靜態紫外/可見光吸收光譜 80 4.2.2螢光分光光譜 81 4.2.3衰減全反射式傅氏轉換紅外吸收光譜 82 4.2.4穿透式電子顯微鏡影像 82 4.3 螢光衰減動力學之數據處理 83 4.3.1色胺酸單體與CAPnWN螢光衰減之數據處理 83 4.3.2修飾於金奈米粒子表面的CAPnWN螢光衰減之數據處理 84 4.4 金奈米粒子對色胺酸螢光放光之作用機制 85 4.4.1能量轉移 85 4.4.2電子轉移 86 參考文獻 112 第五章 結論 116

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