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研究生:徐婉柔
研究生(外文):Wan-Jou Hsu
論文名稱:金奈米粒子表面胰蛋白修飾的尺寸效應與酵素活性之探討
論文名稱(外文):The size effect and enzymatic activity of gold nanoparticles conjugated with trypsin
指導教授:葉晨聖
指導教授(外文):Chen-Sheng Yeh
學位類別:碩士
校院名稱:國立成功大學
系所名稱: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:2004
畢業學年度:92
語文別:中文
論文頁數:75
中文關鍵詞:酵素活性尺寸效應胰蛋白金奈米粒子
外文關鍵詞:enzymatic activitysize effecttrypsingold nanoparticles
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  本篇論文中將胰蛋白修飾在金奈米粒子的表面,觀察胰蛋白修飾後的酵素活性表現情形;另外,因為奈米粒子隨著其粒徑大小的變化也會展現岀不同的性質而引起了廣泛的研究興趣,所以一開始我們在實驗中也嘗試改變金奈米粒子的粒徑,來觀察奈米粒子粒徑變化對於胰蛋白酵素活性的影響。然而,我們進ㄧ步在實驗過程中發現,緩衝溶液(Tris-HCl)的濃度會影響胰蛋白吸附在金奈米粒子表面的數量,因此我們探討不同的緩衝溶液濃度與胰蛋白吸附量以及酵素活  TEM 影像、螢光光譜及DLS 分析確認胰蛋白已修飾在金奈米粒子的表面,並且是以multilayer 的結構存在,由酵素動力學實驗結果顯示出下列幾點性質:(1)將胰蛋白修飾在金奈米粒子表面,會降低其酵素活性。在13 nm 金奈米粒子系統下,緩衝溶液濃度10mM 為例,酵素活性由原本的27.2 product/s•per Trypsin 降低為10.8
product/s•per Trypsin(2)當所修飾的金奈米粒子粒徑由13 nm 換成19 nm 時,胰蛋白的酵素活性反而由8.7 product/s•per Trypsin 降低為5.3 product/s•per Trypsin(3)當緩衝溶液濃度降低時,胰蛋白的吸附量上升,其酵素活性卻隨著吸附量的上升而降低。例如:在13 nm金奈米粒子系統下,當緩衝溶液濃度由10 mM 降低為0.05 mM 時,吸附量由33.0 %上升到50.7 %,酵素活性卻由10.8 product/s•per Trypsin 降低為0.2 product/s•per Trypsin。而導致胰蛋白酵素活性降低的原因,我們推測可能是因為胰蛋白與金奈米粒子間的作用力、multilayer 的結構所造成的立體障礙因素以及奈米粒子粒徑變大時導致胰蛋白構形上的改變或破壞等因素,都將會在本論文中詳細討論。
  In this thesis, immobilization of trypsin on gold nanoparticles was studied to observe the enzymatic activity of gold nanoparticles conjugated with Trypsin. Two sizes(13 and 19 nm)of gold nanoparticles were closen to investigate the size dependence of the enzymatic activity.It was found that the concentration of the buffer solution strongly affected trypsin adsorption and conjugated activity. TEM, fluorescence spectroscopy and dynamic light scattering measurements were employed
to characterize Au/trypsin conjugates. On the basis of the experimental analysis, the following results were attained:(1)the immobilization of trypsin on the gold nanoparticles resulted in decreasing its enzymatic activity, for example, the enzymatic activity decreased from 27.2 product/s•per trypsin to 10.8 product/s•per trypsin using 13 nm gold nanoparticles in 10 mM tris-HCl buffer solution;(2)the increase of the nanoparticle sizes(from 13 nm to 19 nm)accompanied with the decrease of the enzymatic activity ( from 8.7 product/s•per trypsin to 5.3 product/s•per trypsin);(3)the decrease of the buffer concentration resulted in the increase of the trypsin adsorption, but the decrease of the enzymatic activity. For example, the quality of the trypsin adsorption increased from 33.0 % to 50.7 % and the enzymatic activity decreased from 10.8 product/s•per trypsin to 0.2 product/s•per trypsin while the concentrations of the buffer solution decreased from 10 mM to 0.05 mM for 13 nm gold nanoparticles. The resulting
pertaining to enzymatic activity could be interpreted in terms of the interaction of the nanoparticles with the trypsin.
目錄..................................................Ⅰ
中文摘要……………………………………………………………Ⅰ
英文摘要……………………………………………………………Ⅱ
目錄…………………………………………………………………Ⅳ
表目錄………………………………………………………………Ⅵ
圖目錄………………………………………………………………Ⅶ
1-1 漫談奈米粒子………………………………………………1
1-2 奈米粒子的性質……………………………………………3
1-2-1 表面積效應……………………………………………..3
1-2-2 小尺寸效應……………………………………………..5
1-3 奈米粒子之基本理論………………………………………8
1-3-1 影響奈米粒子穩定度的因素…………………………..8
1-3-2 金屬奈米粒子的表面電漿共振……………………….13
1-4 奈米粒子製備的方法…………………………………….18
1-4-1 氧化還原方法………………………………………….18
1-4-2 超音波還原法………………………………………….19
1-4-3 生物方法製備奈米粒子……………………………….19
1-5 酵素固定化的發展與應用(enzyme immobilization)…21
1-6 胰蛋白(Trypsin)之基本簡介………………………..24

第二章胰蛋白修飾金奈米粒子的研究
2-1 研究動機…………………………………………………….36
2-2 實驗藥品與儀器…………………………………………….37
2-3 實驗步驟…………………………………………………….39
2-3-1 13 nm 金奈米粒子之製備……………………………...39
2-3-2 19 nm 金奈米粒子之製備……………………………...39
2-3-3 金奈米粒子之表面胰蛋白修飾…………………….....40
2-3-4 酵素動力學與活性測試………………………………...40

第三章結果與討論
3-1 TEM 分析…………………………………………………...45
3-2 螢光光譜分析……………………………………………….45
3-3 DLS 分析…………………………………………………….46
3-4 酵素動力學分析…………………………………………….47
3-5 討論………………………………………………………...52
3-6 結論………………………………………………………...55
參考文獻………………………………………………………...66
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