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研究生:翁茂&;#23791;
研究生(外文):Weng, Mao-Feng
論文名稱:螢光奈米鑽石的特性和生物應用:專一性、光毒性和能量轉移研究
論文名稱(外文):Characterizations and biological applications of fluorescence nanodiamonds: specifically targeting, phototoxicity, and energy transfer
指導教授:王念夏江素玉
指導教授(外文):Wang, Niann-ShiahChiang, Su-Yu
學位類別:碩士
校院名稱:國立交通大學
系所名稱:應用化學系碩博士班
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:98
中文關鍵詞:螢光奈米鑽石專一性標靶影像光熱治療螢光共振能量轉移
外文關鍵詞:fluorescence nanodiamondsspecific targeted imagingphotothermal therapyfluorescence resonance energy transfer
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近來奈米鑽石在生物醫學上的應用極具潛力,因為螢光奈米鑽石具有良好的光學特性和生物相容性。本研究主要分為三部份:(1)將140 nm紅光奈米鑽石製作成專一性的探針,利用螢光影像研究運鐵蛋白和海拉細胞膜表面受體的作用力;(2)探討不同表面官能基40 nm和140 nm奈米鑽石對胞吞作用的影響,以及觀察奈米鑽石大量進入細胞後的毒性,並利用光熱效應選擇性殺死癌細胞;(3)首次探討綠光和紅光奈米鑽石間是否有螢光共振能量轉移,以拓展生物醫學上的應用。實驗結果顯示,透過運鐵蛋白和受體的作用力,螢光奈米鑽石成功變成專一性的探針並進入細胞,其螢光光譜和螢光生命期特性亦不受外在環境影響。利用流式細胞儀的分析,發現螢光奈米鑽石-運鐵蛋白複合物的胞吞作用效率,約是胺化奈米鑽石的2倍。奈米鑽石有效率的進入細胞後,我們觀察到細胞的生長速度變慢。此外,在532 nm的雷射照射下,專一性進入細胞的奈米鑽石可產生光熱效應,選擇性將被標靶的癌細胞殺死,使用的照射能量是低於殺死未被標靶細胞所需能量的二分之一。綠光奈米鑽石的螢光範圍,和紅光奈米鑽石的吸收範圍有良好的重疊,可形成很好的螢光共振能量轉移配對。我們分別將抗體和抗原固定在綠光和紅光奈米鑽石表面,利用抗原-抗體作用力相結合,其螢光共振能量轉移的效率約是18 %。本研究結果顯示螢光奈米鑽石在生醫應用的可能性,有潛力變成專一性探針和扮演成光熱治療試劑去殺死癌細胞。
Recently, fluorescent nanodiamonds (FNDs) have attracted considerable attention as optical probes in biological and medicinal applications because of their special optical properties and great biocompatibility. In this study, we investigated FNDs as specifically targeted probes to image the interactions of transferrins (Tf) with transferrin receptors (TfR) on HeLa cells, the mechanisms involved in the cellular uptake of surface-modified FNDs, the effects of particle size on the specificity of cellular uptake, and evaluated their cytotoxicity and phototoxicity following particle internalization, and the efficiency of fluorescence resonance energy transfer (FERT) between green and red fluorescent nanodiamonds (gFND and rFND). With Tf immobilized on the surface of rFNDs, we demonstrated that rFND-Tf bioconjugates successfully targeted HeLa cells through Tf-TfR interactions and the surface effects of chemical interactions on the emission of rFND are negligible. Through an analysis of the efficiency of internalized rFND-Tf and rFND-NH2 by flow cytometry, the endocytosis of rFND-Tf particles was highly effective and nearly twice as efficient as that of rFND-NH2 particles. The cytotoxicity of internalized particles resulted in decreasing the rate of proliferation of rFND-treated cells. An examination of the photothermal effect of internalized rFND-Tf particles under irradiation using a 532 nm laser revealed that the rFND-treated cells could be killed selectively using less than half the energy required for untreated cells. gFND and rFND could be a proper FRET pair because the emission of gFND overlaps well with the absorption of rFND. With antibody and antigen immobilized on the surface of gFND and rFND, we observed that the efficiency of FRET was 18 % after the combination of gFND and rFND through antigen-antibody interactions. Our results demonstrated that bioconjugated FNDs could be highly effective in the targeting of cancer cells for fluorescence imaging and photokilling with a minimum of collateral cell damage.
中文摘要 i
英文摘要 ii
誌謝 iv
目錄 v
圖目錄 viii
表目錄 xiv
第一章 緒論 1
1-1 螢光奈米鑽石 1
1-2 研究動機和方法 5

第二章 實驗技術和步驟 13
2-1 實驗原理 13
2-1-1 范氏加速器 13
2-1-2 共焦雷射掃描螢光顯微術 13
2-1-3 螢光生命期影像顯微術 14
2-1-4 螢光共振能量轉移 15
2-1-5 流式細胞儀 16
2-1-6 動態光散射儀 17
2-1-7 界面電位 18
2-1-8 細胞活性測試-MTT檢驗 18
2-2 實驗系統 19
2-2-1 共焦雷射掃描螢光顯微鏡 19
2-2-2 螢光生命期顯微鏡 19
2-2-3 拉曼和螢光光譜儀 20
2-3 實驗步驟 20
2-3-1 螢光奈米鑽石的製備和表面修飾 20
2-3-2 細胞培養和專一性標靶 22
2-3-3 細胞螢光影像 23
2-3-4 流式細胞儀的樣品處理 23
2-3-5 螢光奈米鑽石的細胞毒性 24
2-3-6 螢光奈米鑽石的光毒性 24
2-3-7 螢光共振能量轉移 25

第三章 紅光奈米鑽石的螢光特性和專一性標靶影像應用 37
3-1 奈米鑽石螢光特性 37
3-2 專一性標靶鑑定 40
3-3 結論 42

第四章 紅光奈米鑽石的胞吞作用和毒性研究 54
4-1 表面性質鑑定 54
4-2 胞吞作用細胞螢光影像 55
4-3 胞吞作用效率分析 57
4-4 奈米鑽石細胞毒性 60
4-5 奈米鑽石光毒性 63
4-6 拉曼光譜 65
4-7 結論 67
第五章 奈米鑽石的螢光共振能量轉移 84
5-1 綠光奈米鑽石 84
5-2 螢光共振能量轉移 84
5-3 結論 87

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