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研究生:劉孟學
研究生(外文):Meng-Hsueh Liu
論文名稱:多醣-奈米金複合物之製備、光學性質與對巨噬細胞的影響
論文名稱(外文):Preparation and optical properties of polysaccharide-gold nanoparticles and their effects on marcophages
指導教授:賴鳳羲
指導教授(外文):Phoency Lai
學位類別:碩士
校院名稱:靜宜大學
系所名稱:食品營養研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:112
中文關鍵詞:半胱胺酸奈米金粒子多醣免疫調節巨噬細胞
外文關鍵詞:immunomodulationmacrophagecysteinepolysaccharidegold nanoparticles
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本研究旨在利用檸檬酸鈉當作還原劑,將四氯金酸還原成奈米金粒子,使其粒徑達到 15 與 20 nm 而形成紅色膠狀金溶液,再經半胱胺酸 (cysteine) 修飾金奈米粒子表面使其親水化。粒子初步鑑定及大小藉由 UV/Vis 吸收光譜、掃描式電子顯微法 (scanning electron microscopy, SEM) 及雷射粒徑分析儀 (Dynamic light scattering, DLS) 來觀察,發現奈米金產物在 520 nm 下有一特徵性最大吸收峰且呈球型。比較離心轉速 (8810、16100 xg) 收集沉降奈米金粒子,發現 8810 xg比 16100 xg 較適合收集安定的奈米金粒子。藉由拉曼光譜 (Raman spectrum) 及傅立葉轉換紅外線光譜 (fourier transform infrared spectroscopy, FTIR) 鑑定,發現cysteine 修飾上奈米金在波數 2550 cm-1 下的硫氫基 (S-H) 吸收峰,會消失代表 cysteine 分子上的硫與奈米金核發生硫金共價鍵結 (S-Au) 。經由 UV/Vis 吸收光譜與公式計算,cysteine 修飾之 15 nm 與 20 nm 奈米金回收率分別達到 65.0% 及 32.7%。15 nm 奈米金溶液在 pH 值為 11.5 時,可將多醣上的羥基 (–OH) 與奈米金複合物外端上的羧基 (–COOH) 發生酯化反應 (esterification),產生化學性鍵結。巨噬細胞 (Raw 264.7) 實驗結果得知,單純多醣及多醣-奈米金複合物 (除 xylan 組別外) 可促進 Raw 264.7 生長與刺激分泌細胞激素,包括腫瘤壞死因子 (tumor necrosis factor-α, TNF-α)、介白素-6 (interleukin-6, IL-6) 及一氧化氮 (nitric oxide, NO),尤其以蒟蒻多醣高分子量區分物 (Mw > 10 kDa) 及三仙膠-奈米金複合物最顯著。奈米金和多醣-奈米金複合物具有抑制 LPS 引起 RAW 264.7 產生發炎有關的細胞激素 (IL-6) 的效果。綜合上述,奈米金與多醣-奈米金複合物具有潛在的免疫調節活性。
This study was to preapare a colloidal solution of gold nanoparticles (GNPs) of 15 and 20 nm in diameter synthesized by reduction of HAuCl4 with citrate, followed by using cysteine (Cys) to modify GNPs. The resultant GNPs were characterized by UV/Vis absorption spectra, scanning electron microscopy and dynamic light scattering. The resultant nano-gold aqueous solution showed a maximal absorbance at ( λmax) 520 nm and spheroidal. Comparing two centrifugal rotational speeds (8810,16100 xg) collected GNPs sediments. 8810 xg was better than 16100 xg to get a stable GNPs. Identification of cysteine-capped GNPs by Raman spectroscopy and fourier transform infrared spectroscopy, absorption peak at 2550 cm-1. The disappearance of mercapto group (S-H) indicated the covalent bond formation between sulphur and gold, which was in agreement with FT-IR data. UV/Vis absorption spectra and formula computation, the recovery of cysteine-capped were 65.0% and 32.7%, respectively. By adjusting pH value to 11.5, 15 nm and 20 nm GNPs (Cys-GNPs) of the hydroxyl group (-OH) polysaccharides appeared to bind to the carboxyl group (-COOH) of cysteine-capped GNPs esterification. The in vitro results on macrophage (RAW 264.7) indicate, that polysaccharides and polysaccharide-Cys-GNPs (except for xylan) stimulated RAW 264.7 cells to secrete cytokines, including tumor necrosis factor-α (TNF-α) , interleukin-6 (IL-6) and nitric oxide (NO), especially konjac mannan (MW>10 kDa) and xanthan gum. GNPs and polysaccharide-Cys-GNPs showed an inhibition effect on LPS-induced IL-6 secretion of RAW 264.7. In conclusion, gold nanoparticles and polysaccharide-gold nanoparticles have the potential anti-inflammatory activity.
目 錄
頁 次
中文摘要 ……………………………………………………………………………Ⅰ
英文摘要 ……………………………………………………………………………Ⅱ
謝誌 …………………………………………………………………………………Ⅲ
目錄 …………………………………………………………………………………Ⅳ
圖目錄 ………………………………………………………………………………Ⅵ
表目錄 ………………………………………………………………………………Ⅷ
名詞縮寫 ……………………………………………………………………………Ⅸ
樣品目錄 ……………………………………………………………………………Ⅹ

第一章 前言 ………………………………………………………………… 1
第二章 文獻回顧 …………………………………………………………… 3
第一節 奈米材料之簡介 …………………………………………………… 3
第二節 奈米金粒子之介紹 ………………………………………………… 7
第三節 半胱胺酸( cysteine )之簡介 ……………………………………… 17
第四節 食品多醣膠質之簡介 …………………………………………… 21
第五節 免疫反應與巨噬細胞 …………………………………………… 29
第三章 材料與方法 ……………………………………………………… 34
第一節 實驗流程 ………………………………………………………… 34
第二節 實驗材料 ………………………………………………………… 42
第三節 樣品製備方法 …………………………………………………… 45
第四節 粒子表面型態測量與濃度計算 ………………………………… 49
第五節 細胞培養與奈米金複合物對免疫細胞增生試驗 ……………… 54
第六節 Raw 264.7 cell 激素的測定 ……………………………………… 56
第七節 Raw 264.7 cell 之 NO 的測定 …………………………………… 62
第八節 分析統計 ………………………………………………………… 63
第四章 結果與討論 ………………………………………………………… 64
第一節 奈米金之製備與濃度計算 ………………………………………… 64
一. 不同粒徑奈米金製備 ……………………………………………………64
二. 初步計算奈米金溶液濃度 ………………………………………………66
三. 估算奈米金粒子表面原子數 ……………………………………………66
第二節 奈米金微粒粒徑篩分 ……………………………………………… 67
第三節 Cysteine 修飾奈米金粒子之較適製備條件 ……………………… 70
一. 奈米粒子經cysteine修飾後光譜的改變 …………………………… 70
二. 改質前後回收率計算與粒徑大小改變 …………………………………75
三. 奈米金粒子表面官能基鑑定與粒徑分佈 ………………………………77
第四節 多醣-奈米金複合物活化巨噬細胞(Raw 264.7 cell)的效果 ……… 84
第五章 結論 ………………………………………………………………… 89
第六章 參考文獻 …………………………………………………………… 91








圖目錄
頁 次
圖一 金屬粒子在一電場下的極化示意圖 …………………………… 6
圖二 水相中粒徑9,22,48,及99 nm 之
最大吸收峰依序為517,521,533 及575 nm………………… 6
圖三 金奈米粒子組成示意圖 ………………………………………… 8
圖四 12 nm 金奈米溶液添加 1 M NaCl 混合六分鐘後(聚集)
之可見光譜圖 …………………………………………………… 9
圖五 不同粒徑金奈米粒子的UV/Vis 吸收光譜圖 ………………… 12
圖六 光散射於不同形狀、大小與組成的奈米粒子溶液所呈現的顏色 12
圖七 敘述 NRGNPs 添加帶中性硫醇分子及正電性硫醇分子
產生 FRET 及聚集之機制 …………………………………… 15
圖八 NRGNPs 修飾不同帶電性之硫醇分子後的外觀顏色及
穿透式電子顯微鏡圖。(a) 負電性硫醇分子、(b) 中性
硫醇分子、(c) 正電性硫醇分子……………………………… 16
圖九 半胱胺酸之結構 ………………………………………………… 17
圖十 雷立和拉曼散射的來源 ………………………………………… 20
圖十一 阿拉伯膠之基本結構…………………………………………… 24
圖十二 纖維素 (cellulose) 及羧甲基纖維素之分子結構……………… 25
圖十三 蒟蒻葡萄糖聚甘露醣 (konjac glucomannan) 之結構…………… 26
圖十四 三仙膠基本結構 ………………………………………………… 27
圖十五 硬木 (hard wood) 聚木醣結構 ……………………………… 28
圖十六 軟木 (soft wood) 聚木醣結構 ………………………………… 28
圖十七 先天性免疫反應與應變性免疫反應機制 ………………………… 30
圖十八 LPS造成體內免疫系統的活化 ………………………………… 33
圖十九 LPS 使巨噬細胞產生細胞激素 ………………………………… 33
圖二十 迴流加熱裝置圖 ………………………………………………… 46
圖二十一 奈米金微粒篩分流程圖 ………………………………………… 47
圖二十二 JOEL JSM-6700F場發射掃描式電子顯微鏡 …………………… 50
圖二十三 LabRAM HR800 拉曼光譜儀 …………………………………… 51
圖二十四 15 nm及20 nm之金奈米粒子吸收光譜圖 ………………………64
圖二十五 奈米金粒子於不同貯存時間吸收光譜圖 ……………………… 65
圖二十六 15 nm 金奈米粒子於不同離心轉速收集之可見光譜圖………… 67
圖二十七 20 nm 金奈米粒子於不同離心轉速收集之可見光譜圖………… 68
圖二十八 15 nm 及20 nm GNPs經8810 xg 及16100 xg 離心
後之SEM影像圖 ………………………………………………… 69
圖二十九 15 nm GNPs 之雷射粒徑分佈圖 ………………………………… 69
圖三十 奈米金粒子經半胱胺酸修飾後外觀圖 ………………………… 70
圖三十一 15 GNP (M) 經cysteine修飾後之光譜圖 ……………………… 71
圖三十二 15 GNP (MS) 經cysteine修飾後之光譜圖 …………………… 72
圖三十三 20 GNP (M) 經cysteine修飾後之光譜圖 ……………………… 73
圖三十四 20 GNP (MS) 經cysteine修飾後之光譜圖……………………… 74
圖三十五 金奈米粒子親水化改質前後的吸收光譜比較…………………… 75
圖三十六 不同粒徑之奈米金粒子經10 mM cysteine改質之SEM影像圖 76
圖三十七 15 nm GNPs經10 mM cysteine 改質後之DLS粒徑分佈圖…… 76
圖三十八 半胱胺酸與半胱胺酸修飾奈米金之紅外線光譜圖…………… 77
圖三十九 半胱胺酸與半胱胺酸修飾奈米金之拉曼光譜圖 ……………… 78
圖四十 多醣修飾奈米金複合物之外觀圖 ……………………………… 79
圖四十一 奈米金修飾後與多醣鍵結之紅外線光譜圖 …………………… 80
圖四十二 15 nm 奈米金修飾後與多醣鍵結之 DLS 粒徑分佈圖 ……… 81
圖四十三 多醣、奈米金與修飾後接上多醣對巨噬細胞(Raw 264.7 cell)
增生率的影響 …………………………………………………… 86
圖四十四 多醣、奈米金與修飾後接上多醣對巨噬細胞(Raw 264.7 cell)
NO的生成 ………………………………………………………… 87
圖四十五 多醣、奈米金與修飾後接上多醣對巨噬細胞(Raw 264.7 cell)
作用生成細胞激素之效果 ……………………………………… 88
表目錄
頁 次表一 奈米微粒尺寸與表面原子數之關係 ……………………………… 4
表二 常見合成奈米金粒子的方法 ……………………………………… 8
表三 金屬分類表週期表……………………………………………………10
表四 可見光部份的波長與其所顯示的各種顏色及其互補色 ………… 13
表五 FTIR 吸收訊號與相對官能基之相關簡表 ……………………… 18
表六 具特定生理活性的食品多醣及其成份 ………………………… 22
表七 具抗氧化活性的食品多醣 ……………………………………… 23
表八 先天性和應變性免疫中細胞激素的特性比較 …………………… 31
表九 離心力換算表 …………………………………………………… 47
表十 奈米金溶液與修飾半胱胺酸的製備條件 ………………………… 78
表十一 多醣與奈米金複合物進行物理性錯合 …………………………… 82
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