臺灣博碩士論文加值系統

本研究以基因轉殖大腸桿菌進行CdS奈米粒子的製備，研究內容結合生物科技以及奈米技術兩個領域。CdS為擁有直接能隙的II-V半導體材料，粒徑小於6 nm以下時，有明顯的量子侷限效應，被視為新一代的LED發光材料。除此之外，CdS奈米粒子應用於各類感測器以及生物螢光標定中的研究也相當的多。
本研究首次利用基因工程的手法，將gshA及gshB基因片段以重組載體送入細胞中，分別表現glutathione合成酵素γ-GCS及GS，藉此促進大腸桿菌中glutathione的含量，來量產CdS奈米粒子。GSH分子為生物合成CdS奈米粒子之關鍵，其含量之增加有助於細胞生成CdS粒子。我們證實，大腸桿菌ABLE C生成之CdS奈米粒子粒徑介於2~5 nm之間。經過基因轉殖的菌株，細胞中GS合成酵素含量增加，細胞中CdS粒子產量有顯著提升。
粒子特性方面，基因轉殖大腸桿菌所生產的CdS奈米粒子由於粒子表面有較多的GSH包覆，因此在水溶液中分散性更為提升，而重組蛋白的產生對所生成CdS的粒徑無太大影響。在此我們證明了以大腸桿菌生產CdS奈米粒子的可行性，透過基因轉殖細胞能更近一步增進產量和改善產物性質。在實驗過程中，我們也證實了GSH含量的多寡，確實引響細胞生產CdS的能力，對於此系統背後生理機制的釐清也有所助益。

Cadmium sulfide nanoparticles were synthesized intracellularly in gene modified Escherichia coli ABLE C. The nanoparticles, a known semiconducting material, which have unique optical and electronic properties, have potential for application in the emerging field of nanoelectronics. To produce nanomaterial cheaply and efficiently, biological methods of synthesis are being explored. Schizosaccharomyces pombe were found having the capacity to synthesize CdS nanoparticles intracellularly in 1983. Recently, in 2004, Sweeney found that E. coli also have ability to synthesize CdS nanoparticles. Morever, in his study, he pointed out that the cellular glutathione can enhance the formation of nanoparticles. Inspried by Sweeney, we constructed a gene modified E. coli ABLE C which could use for glutathione overproduction and attempt to figure out how this parameter can enhance the synthesis of nanoparticles within bacterial cells. Our results showed that nanoparticles biosynthesis increased about 1.6 –fold in gene modified cells compared to wild type cells. HRTEM data showed that the nanoparticles had a Wurtzite hexagonal lattice structure and most of the nanoparticles were in the size range of 2-5 nm. We also observed that nanoparticles synthesized in gene modified cells were dispersed better in water than these in wild type cells. The well dispersion behavior was due to more glutathione coated on the nanoparticles. To our best knowledge, this is the first report using genetic modification to enhance biosynthsis of CdS nanoparticles and to unravel the siginificance of glutathione in this procedure. By understanding parameters of nanoparticles synthesis, it might be possible to modulate the properties of biosynthesized nanopartilces, such as size, shae, and crystal structure.

目錄 I
圖目錄 .V
表目錄.VI
第1章 第一章：緒論 1
第2章 第二章：文獻回顧 2
2-1 ：半導體發光材料 2
2-2 ：奈米材料之簡介 5
2-2-1 ：奈米粒子 5
2-2-2 ：奈米粒子的製備方式 6
2-2-3 ：奈米科技之發展 7
2-3 硫化鎘(CdS)簡介 8
2-3-1 CdS性質 8
2-3-2 CdS的應用 8
2-3-3 CdS奈米粒子的製備方式 9
2-4 基因轉殖大腸桿菌製備CdS奈米粒子 12
2-4-1 以生物系統製備CdS奈米粒子 12
2-4-2 大腸桿菌表現系統 14
2-4-3 Glutathione (GSH) 15
2-5 研究動機及目的 16
第3章 第三章：實驗材料與方法 18
3-1 實驗材料 18
3-1-1 菌株 18
3-1-2 表現載體 18
3-1-3 藥品與酵素 18
3-2 大腸桿菌重組蛋白表現系統 18
3-2-1 大腸桿菌培養 18
3-2-2 表現載體之改良 19
3-2-3 連鎖聚合酶反應(PCR reaction) 19
3-2-4 轉型反應(Transformation) 20
3-2-5 大腸桿菌質體之少量抽取 21
3-2-6 重組質體之確認 21
3-2-7 重組蛋白之誘導表現 21
3-2-8 蛋白質電泳 22
3-2-9 蛋白質定量分析 22
3-2-10 Glutathione (GSH)之定量 23
3-2-11 細胞培養基成分之調整 24
3-2-12 細胞於重金屬環境壓力下的GSH表現反應 24
3-3 硫化鎘奈米粒子合成反應與分析 25
3-3-1 CdS奈米粒子生成反應 25
3-3-2 細胞破碎處理 25
3-3-3 CdS奈米粒子定量 26
3-3-4 穿透式電子顯微鏡觀察 27
3-3-5 高解析穿透式電子顯微鏡 27
第4章 第四章：實驗結果與討論 31
4-1 重組大腸桿菌之建構 31
4-2 轉殖基因之表現 32
4-2-1 轉型後細胞生長曲線 32
4-2-2 SDS-PAGE蛋白質電泳分析重組蛋白之表現 33
4-3 GSH與細胞生成CdS能力之探討 35
4-3-1 重金屬環境下GSH合成途徑之改變 35
4-3-2 CdS奈米粒子生成量之提升 36
4-3-3 培養基添加物對GSH含量之影響 37
4-3-4 GS酵素表現對細胞內GSH含量之影響 39
4-3-5 GS酵素表現對細胞合成CdS奈米粒子之影響 40
4-4 CdS奈米粒子之觀察 50
4-4-1 穿透式電子顯微鏡(TEM)觀察 50
4-4-2 高解析穿透式電子顯微鏡(HRTEM)觀察 51
4-4-3 能量分散光譜儀(Energy Dispersive X-ray Analysis, EDX)分析 52
4-4-4 綜合討論 52
第5章 第五章：結論 57
第6章 第六章：未來展望 59
6-1 CdS奈米粒子性質之探討 59
6-1-1 CdS奈米粒子之分離 59
6-1-2 CdS奈米粒子表面鍵結蛋白之分析 59
6-1-3 CdS奈米粒子光學性質探討 60
6-2 CdS奈米粒子應用性評估 61
第7章 參考文獻 63

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