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研究生:陳怡云
研究生(外文):Yi-Yun Chen
論文名稱:金奈米顆粒表面改質應用於細胞觀測之研究
論文名稱(外文):Surface modification of gold nanoparticles for the application of cellular imaging
指導教授:廖駿偉蔡曉雯蔡曉雯引用關係
指導教授(外文):Jiunn-Woei LiawShiau-Wen Tsai
學位類別:碩士
校院名稱:長庚大學
系所名稱:生化與生醫工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:71
中文關鍵詞:金奈米粒子葉酸受器-轉介的內噬作用雷射掃描式共軛焦顯微鏡
外文關鍵詞:gold nanoparticlesfolic acidreceptor-mediated endocytosisLaser scanningconfocal microscopy
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此研究目標主要為利用金奈米粒子之特殊光學性質,結合配體-受體(ligand- receptor)的專一性,並且利用雷射掃描式共軛焦顯微鏡觀察,比較正常細胞與腫瘤細胞對於表面修飾及未修飾葉酸分子的金奈米粒子吞噬狀況。實驗以種晶促進長成法製備球型金奈米粒子以及各種長短軸比(aspect ratio)之金奈米桿,另外,利用傅利葉紅外光儀及1H-核磁共振光譜儀,確認葉酸鍵結於金奈米粒子的表面。接著我們使用表面未修飾葉酸的金奈米粒子與乳癌細胞以及正常乳房細胞共培養30分鐘,發現正常乳房細胞攝入的奈米粒子量會多於乳癌細胞,而且與細胞共培養的金奈米粒子濃度愈高、時間愈長,單位細胞攝入金奈米粒子量愈多。另外,將經過葉酸修飾的金奈米粒子與乳癌及正常乳房細胞共培養5分鐘,其共軛焦的細胞影像有極強的表現,可推論經過葉酸修飾的金奈米粒子對於表面帶有葉酸受體的細胞而言,能經由專一性鍵結的幫助,提升細胞吞噬金奈米粒子的速度。經由穿透式電子顯微鏡的觀察,證實金奈米粒子進入到細胞質內,而不只是附著於細胞膜表面。故經葉酸表面修飾的金奈米粒子,未來可作為生物標定物(biomaker)的應用。
The purpose of this study is twofold; one is to improve the uptake of gold nanoparticles by specific cells with hFR (folate receptor) by surface-modifying these gold nanoparticles with folic acid, and the other is to investigate the optical response of gold nanoparticles by using laser scanning confocal microscopy for the application of cellular imaging. For our experiments, we use seed-mediated growth method to synthesize spherical gold nanoparticles and different aspect-ratio gold nanorods. These gold nanoparticles are surface-modified by alkanethiols firstly, and then are linked with folic acid. By using Fourier transom infrared spectroscopy (FTIR) and 1H-NMR spectroscopy, these surface-modified gold nanoparticles are tested, and the functional groups of folic acid are measured to verify the conjugation of folic acid with these gold nanoparticles. Two kinds of cells are used for test; one is the normal human breast cells (H184B5F5), and the other is the human breast tumor cells (435s). Both have high expression of hFR at their membranes. We incubate these cells with different solutions containing different optical densities of gold nanoparticles with and without folic acid. The nucleuses of these cells are also stained by PI. Using laser scanning confocal microscopy, the scattering expression of these gold nanoaprticles and the fluorescence of PI can be clearly observed simultaneously for different wavelength lasers (458nm, 488nm, 514nm, 561nm, and 633nm). Since the intensity of the scattering light is proportional to the amount of gold nanoparticles, the cellular images of laser scanning confocal microscopy can be used to indicate the amount of gold nanoparticles qualitatively. The images show that gold nanoparticles are distributed in the cytoplasm. In addition, the results show that the higher the concentration of gold nanoparticles (with and without folic acid) in the solution and the longer the co-culture time of cells with the solution, the more the uptake of gold nanoparticles by the cells. Furthermore, the uptake speed of gold nanoparticles coated with folic acid is increased a lot, compared to those without folic acid, for both H184B5F5 and 435s cells. TEM images also show that these gold nanoparticles are aggregated in the vesicles in the cytoplasm, rather than attached on the membrane. This phenomenon illustrates gold nanoparticles are taken up by cells through the endocytosis process.
目 錄
指導教授推薦書
口試委員審定書
授權書 iii
誌謝 iv
中文摘要 v
Abstract vi
第一章 緒論 1
1.1 研究背景 1
1.2 研究動機與目的 6
第二章 文獻回顧 7
2.1 金奈米粒子的介紹 7
2.2 金奈米粒子的合成 9
2.3 奈米粒子於生醫上的應用 12
第三章 實驗方法 20
3.1 藥品及儀器設備 20
3.2 金奈米粒子的製備 22
3.3 金奈米粒子的表面修飾 24
3.4 金奈米粒子之特性分析 27
3.4.1 紫外光/可見光/近紅外光(UV/VIS Near-IR)光譜分析 27
3.4.2 穿透式電子顯微鏡 (Transmission electron microscopy,
TEM)觀察 28
3.4.3 傅利葉紅外光光譜儀 (Fourier Transform Infrared, FT-IR)
及核磁共振儀(1H-NMR)量測 28
3.5 動物細胞培養 29
3.5.1 細胞毒性測試 29
3.5.2 以雷射掃描式共軛焦顯微鏡(Laser scanning confocal
microscopy)觀察金奈米粒子於細胞胞內之分布狀況 30
3.5.3 動物細胞之細胞核染色 31
3.5.4 穿透式顯微鏡細胞樣品之製備 32
3.5.5 以不同波長雷射觀察螢光染劑PI與金奈米粒子 33
第四章 結果與討論 35
4.1 金奈米粒子的製備 35
4.2 金奈米粒子的表面修飾 36
4.3 細胞毒性測試 37
4.4 細胞吞噬實驗 38
4.5 不同雷射波長觀察螢光染劑PI與金奈米粒子 42
第五章 結論 45
參考文獻 68



圖目錄
圖1.1、不同形狀、大小之金奈米粒子,由左至右長短軸比依序
增加 5
圖1.2、與圖1.1金奈米粒子溶液對照的UV-Vis Near IR光譜圖
5
圖2.1、種晶促進長成法 11
圖2.2、以葉酸標的的奈米微脂粒結構 17
圖2.3、葉酸分子與細胞膜受器結合的胞噬作(Receptor-mediated
endocytosis of folate conjugates)機制 18
圖2.4、葉酸之分子結構 18
圖2.5、癌細胞與正常細胞上葉酸受器(αhFR)的表現程度 19
圖3.1、金奈米粒子製備流程圖 23
圖3.2、葉酸修飾之金奈米粒子結構 25
圖3.3、金奈米粒子的表面修飾流程圖 26
圖3.4、PI的螢光激發(excitation)與放射(emission)圖 34
圖4.1、不同特徵吸收波長之金奈米粒子之UV-Vis NIR 光譜圖 47
圖4.2、穿透式電子顯微鏡觀察結果(x100k) A. 530 nm B.640nm
C. 850nm 之金奈米粒子 48
圖4.3、以2-Mercapto-Ethylamine修飾金奈米粒子之UV-Vis NIR
光譜圖 49
圖4.4、以葉酸修飾金奈米粒子之UV-Vis NIR光譜圖 50
圖4.5、以葉酸修飾金奈米粒子之傅利葉紅外光光譜圖 51
圖4.6、(a)金奈米粒子(b)葉酸之1H-液態核磁共振光譜圖 52
圖4.7、以葉酸分子修飾金奈米粒子之1H-液態核磁共振光譜圖 53
圖4.8、435s乳癌細胞之細胞形態(a)未加入金奈米粒子 (b)與加入
O.D. 0.5未修飾葉酸之金奈米粒子共培養24小時(20x) 54
圖4.9、O.D.值為0.33的未修飾葉酸之金奈米粒子與435s細胞共
培養30分鐘之共軛焦顯微鏡影像(63x)。A-C.細胞型態與
金奈米粒子散射光之重疊影像。D-F.金奈米粒子受到
561nm雷射照射後之散射光 55
圖4.10、O.D.值為0.85的未修飾葉酸之金奈米粒子與435s細胞
共培養30分鐘之共軛焦顯微鏡影像(63x)。A-C.細胞型態與
金奈米粒子散射光之重疊影像。D-F.金奈米粒子受到
561nm雷射照射後之散射光 56
圖4.11、O.D.值為0.33的未修飾葉酸之金奈米粒子與435s細胞
共培養1小時之共軛焦顯微鏡影像(63x)。A-C.細胞型態與
金奈米粒子散射光之重疊影像。D-F.金奈米粒子受到
561nm雷射照射後之散射光 57
圖4.12、O.D.值為0.54的未修飾葉酸之金奈米粒子與H184B5F5
細胞共培養30分鐘之共軛焦顯微鏡影像(63x)。A-C.細胞
型態與金奈米粒子散射光之重疊影像。D-F.金奈米粒子受
到561nm雷射照射後之散射光 58
圖4.13、O.D.值為0.54的未修飾葉酸之金奈米粒子與H184B5F5
細胞共培養1小時之共軛焦顯微鏡影像(63x)。 A-C.細胞
型態與金奈米粒子散射光之重疊影像。D-F.金奈米粒子
受到561nm雷射照射後之散射光 59
圖4.14、O.D.值為0.32的未修飾葉酸之金奈米粒子與435S細胞
共培養30分鐘之共軛焦顯微鏡影像(63x)。A-C.細胞型態
與金奈米粒子散射光之重疊影像。D-F.金奈米粒子受到
561nm雷射照射後之散射光 60
圖4.15、O.D.值為0.37的未修飾葉酸之金奈米粒子與H184B5F5
細胞共培養30分鐘之共軛焦顯微鏡影像(63x)。A-C.細胞
型態與金奈米粒子散射光之重疊影像。D-F.金奈米粒子
受到561nm雷射照射後之散射光 61
圖4.16、O.D.值為0.35的修飾上葉酸分子之金奈米粒子與435s
細胞共培養5分鐘之共軛焦顯微鏡影像(63x)。A-C.細胞
型態與金奈米粒子散射光之重疊影像。D-F.金奈米粒子
受到561nm雷射照射後之散射光 62
圖4.17、O.D.值為0.39的修飾上葉酸分子之金奈米粒子與
H184B5F5細胞共培養5分鐘之顯微影像(63x)。A-C.細胞
型態與金奈米粒子散射光之重疊影像。D-F.金奈米粒子
受到561nm雷射照射後之散射光 63
圖4.18、O.D. 0.5之金奈米粒子與乳癌細胞(435s)共培養24小時後
之穿透式電子顯微鏡觀察結果 (A)x 5k (B)x 40k (C)x 150k
64
圖4.19、O.D.0.5之金奈米粒子與正常乳房細胞(H184B5F5)共培養
24小時後之穿透式電子顯微鏡觀察結果 (A)x5k (B)x50k
(C)x 150k 65
圖4.20、不含金奈米粒子之正常乳房細胞(H184B5F5)經PI
(propidium iodide)染色後受到不同波長雷射照射後之共
軛焦顯微影像(x 63)。A-E依序為短波長至長波長 66
圖4.21、經PI染色後,O.D.0.37未修飾葉酸之530nm金奈米粒子
與正常乳房細胞(H184B5F5)共培養3小時,受到不同波長
雷射照射後之共軛焦顯微影像(x 63)。A-E依序為短波長
至長波長 67









表目錄
表1.1、生物分子之微觀尺度 4
表3.1、不同種類雷射的波長及功率大小 30
表3.2、不同種類雷射照射PI的波長及功率大小 33
表3.3、不同種類雷射照射金奈米粒子的波長及功率大小 33
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