利用蛋白質模板修飾的金奈米團簇(Gold nanoclusters, AuNCs)因具有特殊的光化學性質已經廣泛作為螢光傳感器使用，而酸鹼值、反應時間以及蛋白質模板的差異，會影響金奈米團簇的顆粒尺寸和團聚效果，進而導致不同的螢光放射波長，然而以單一激發表現出雙重放射的螢光探針，因具有不同強度比例的差異可以更準確的定量分析物，已經成為分析感測和生物影像的發展趨勢。
在本篇研究中，使用第六型溶菌酶Lysozyme為蛋白質模板開發出新型雙螢光放射之溶菌酶奈米顆粒包覆金奈米團簇(LysNPs-AuNCs)，以340 nm波長激發於425 nm放射出藍色螢光和820 nm放射出近紅外螢光，不但具有雙螢光的特性且放光波長位於近紅外光波段。由於近紅外光的高穿透性、較不受生物組織自發螢光的干擾和能量較低不易破壞正常細胞，加上金奈米團簇本身具有良好的抗光漂白性、耐鹽性、寬廣的酸鹼值容忍性、抵抗蛋白質水解的能力以及低毒性，因此此篇研究的材料對於生物體內有一大優勢，可以更寬廣運用在各種生物體中。
由於金奈米團簇是利用蛋白質模板所合成，可在蛋白質表面上修飾鏈親和素(Streptavidin)，再將針對癌細胞具有專一性標記的抗體修飾生物素(Biotin)，藉由streptavidin及biotin兩者間高親和力能有效將材料修飾上抗體，最後再利用此抗體標記癌細胞後，以光源激發產生螢光放射的現象，因此可以有效達到癌細胞成像。除此之外，在LysNPs-AuNCs和鏈親和素間修飾上長碳鏈的PEG(Poly ethylene glycol)，不僅能夠增長LysNPs-AuNCs於腫瘤位置的滯留時間，還能有效地降低毒性以達到更準確的腫瘤尺寸以及位置的判斷依據。

Recently, modification of gold nanocluster with different protein template has been developed as biosensor by fluorometric. Different functional modified gold nanocluster exhibits different particle size and optical species due to pH value, reaction time, various proteins and so on. As a result, the emission of the gold nanocluster could be adjusted to apply. However, the facile ratiometric fluorescent probes have become the analysis and cell-image developing trend.
In this study, we report a novel duel- fluorometric LysNPs-AuNCs which modified with type VI Lysozyme as the protein template, and the emission at 425 nm and 820 nm of LysNPs-AuNCs is excited by UV light at 340 nm. The near-infrared characteristic of LysNPs-AuNCs can provide deep imaging penetration, low fluorescence background and less damage of normal cell, and LysNPs-AuNCs is anti-photobleaching, salt tolerance, wide-range of pH, proteolysis and low toxicity can be widely used in the organisms.
The streptavidin could be combined with the surface of LysNPs-AuNCs, and the modification of biotin with antibody which is specifically labeled cancer cells via high affinity between streptavidin and biotin. As a result, the fluorescence of Lys NPs-AuNCs is excited by UV source after the cancer cells are labeled on the antibody so that the imaging of cancer cells can be effectively achieved. Additionally, PEG (polyethylene glycol) modified between LysNPs-AuNCs and streptavidin not only increases the residence time of Lys NPs-AuNCs at the tumor site, but it also decreases the toxicity and achieves a more accurate tumor size and location.

論文審定書 i
論文公開授權書 ii
謝誌 iii
摘要 iv
Abstract v
目錄 vi
圖目錄 ix
表目錄 xii
微波輔助合成近紅外光發光之金奈米團簇並應用於生物體內外 1
1.1 前言 1
1.2 實驗部分 6
1.2.1 實驗藥品 6
1.2.2 儀器設備 9
1.2.3 溶液配製 13
1.2.4 合成溶菌酶奈米顆粒 (LysozymeNPs) 14
1.2.5 合成溶菌酶為模板的金奈米團簇 (Lys-AuNCs) 14
1.2.6 合成溶菌酶奈米顆粒為模板的金奈米團簇 (LysNPs-AuNCs) 15
1.2.7 偵測溫度與pH值 16
1.2.8 偵測鹽類與穀胱甘肽 (GSH) 16
1.2.9 偵測胰蛋白酶 (Trypsin) 與其選擇性 16
1.2.10 單粒子螢光圖像(Single particle image) 17
1.2.11 生物接合 (Bioconjugation) 17
1.2.12 細胞實驗 17
1.2.12.1 細胞實驗樣品前處理 17
1.2.12.2 細胞養殖(cell culture) 18
1.2.12.3 細胞毒性分析 (MTT assay) 19
1.2.12.4 細胞內吞作用 (Endocytosis) 19
1.2.12.5 細胞標記 (Cell labeling) 20
1.2.13 斑馬魚動物實驗 21
1.2.13.1 斑馬魚配魚、孵化、去卵殼 21
1.2.13.2 斑馬魚實驗樣品前處理 22
1.2.13.3 斑馬魚毒性分析 23
1.2.13.4 斑馬魚注射 23
1.2.14 小鼠動物實驗 25
1.2.14.1 小鼠腦部注射 25
1.2.14.2 小鼠腫瘤樣品前處理 25
1.2.14.3 小鼠尾部注射 25
1.3 結果與討論 27
1.3.1 合成並鑑定蛋白質奈米顆粒為模板的金奈米團簇 27
1.3.1.1 溶菌酶奈米顆粒為模板的金奈米團簇 27
1.3.1.2 其他蛋白質奈米顆粒為模板的金奈米團簇 52
1.3.2 對於以蛋白質為模板的金奈米團簇螢光穩定性測試 55
1.3.2.1 對於LysNPs-AuNCs金奈米團簇的螢光穩定度 55
1.3.2.2 對於Lys-AuNCs金奈米團簇的螢光穩定度 66
1.3.3金奈米團簇LysNPs-AuNCs合成最適化條件探討 69
1.3.4 生物體外及體內LysNPs-AuNCs的應用 81
1.3.4.1 LysNPs-AuNCs生物體外及體內的螢光顯影 81
1.3.4.2 LysNPs-AuNCs癌細胞標記的顯影 92
1.4 結論 99
1.5 參考文獻 100

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