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研究生:李牧廸
研究生(外文):Mu-de Li
論文名稱:以金奈米粒子濃縮萃取生物分子結合毛細管電泳進行分析
指導教授:曾韋龍曾韋龍引用關係
指導教授(外文):Wei-lung Tseng
學位類別:碩士
校院名稱:國立中山大學
系所名稱:化學系研究所
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:2009
畢業學年度:97
語文別:中文
論文頁數:88
中文關鍵詞:胺基酸硫醇金奈米粒子毛細管電泳
外文關鍵詞:indoleamineTween 20aminothiolgold nanoparticles
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本篇論文將使用金奈米粒子做為萃取的工具,對水溶液中的生物小分子─胺基酸硫醇(Aminothiols)與吲哚胺類(Indoleamines)─進行濃縮萃取,並利用毛細管電泳分離,成功地提升偵測靈敏度,以及對尿液樣品中的上述生物小分子進行定量。
一、利用非離子型界面活性劑修飾之金奈米粒子選擇性的萃取水溶液中的胺基酸硫醇類小分子並結合毛細管電泳紫外光吸收法進行分析:在在本篇研究中,我們利用非離子型界面活性劑(Tween 20)修飾在金奈米粒子的表面上(Tween 20-capped gold nanoparticles,Tween 20-AuNPs)作為探針,選擇性的從水溶液中萃取欲偵測的胺基酸硫醇類小分子(Aminothiols)。Tween 20分子以非共價性的方式吸附在金奈米粒子的表面上使得金奈米粒子表面上具有堅固及緊密的立體位障,藉由Aminothiols與金奈米粒子表面形成金-硫鍵結,可以針對所欲偵測的Aminothiols做選擇性的萃取。當將Aminothiols加入Tween 20-AuNPs時,會發生聚集的現象,接著將其離心並把上清液移除後,加入高濃度的取代試劑—1,4-二硫蘇糖醇(Dithiothreitol,DTT)—將吸附在金奈米粒子表面上的Aminothiols取代出即完成萃取濃縮的步驟,我們將此萃取技術結合毛細管電泳吸收儀進行偵測,並於毛細管電泳中進行線上濃縮。在最佳的萃取效率及濃縮條件下,麩胱甘肽(Glutathione,GSH)、半胱胺酸(Cysteine,Cys)和同半胱胺酸(Homocysteine,HCys)這三種Aminothiols的偵測極限(LOD)可分別達到 28、554 和 456 nM ,而與傳統的進樣方式相比(未經過 Tween 20-AuNPs 萃取),偵測靈敏度可分別改善 2280、998 和 904 倍。此技術已應用人類尿液樣品中的 GSH 和 HCys 分析,相信未來應能拓展至臨床檢測。
二、利用檸檬酸鈉金奈米粒子萃取吲哚胺類並結合毛細管電泳雷射誘導自發螢光法進行偵測:本篇研究使用檸檬酸鈉金奈米粒子(Citrate-capped gold nanoparticles,Citrate-AuNPs)選擇性地萃取水溶液中吲哚胺類分析物(Indoleamines),如5-hydroxytryptophan(5-HTP)、Tryptophan(Trp)、5-hydroxytryptamine (5-HT)、Tryptamine(TA)與5-hydroxyindoleacetic acid(5-HIAA),隨後再結合毛細管電泳雷射誘導自發螢光法(CE/LINF)對萃取出的Indoleamines分析物進行偵測。由檸檬酸鈉金奈米粒子(Citrate-AuNPs)的消光光譜(Extinction spectra)觀察可得知,將Indoleamines加入後,金奈米粒子發生聚集的現象,主要原因為所加入的Indoleamines會吸附在金奈米粒子表面上,中和了Citrate-AuNPs表面所帶的負電荷,使金奈米粒子靠近產生聚集。其中5-HTP、Trp與5-HIAA是透過其結構中的吲哚環與金奈米粒子表面間之疏水性所形成的凡得瓦力而吸附在金奈米粒子表面上;5-HT與TA則是透過其結構中的胺基與金奈米粒子表面上的檸檬酸鹽(Citrate)形成靜電吸引力而吸附在金奈米粒子表面上。上述的結果證明了Indoleamines會吸附在金奈米粒子表面上達到萃取的效果,隨後只要加入了高濃度的 2-mercaptoethanol(2-ME),能夠將吸附在金奈米粒子表面上的Indoleamines取代出,其原因在於2-ME結構會與金奈米粒子表面形成強而穩固的金-硫鍵結(Au-S bond ),透過此概念,不論是增加金奈米粒子的濃度、金奈米粒子與Indoleamines的反應時間或樣品體積都能有效地增加靈敏度。我們將此技術與不利用金奈米粒子作為萃取探針相比,以金奈米粒子最適當之萃取條件下及結合最佳的毛細管電泳雷射誘導自發螢光法(CE/LINF)分離情況下,可將5-HTP、Trp、TA、5-HT及 5-HIAA的靈敏度分別地提高 48、4077、985、920及4030倍;此外,這項技術也可成功地偵測分析人類尿液樣品中的 TA 與 5-HT。
摘要I
目錄IV
圖目錄VII
表目錄X
縮寫表XI

第一章、利用非離子型界面活性劑修飾之金奈米粒子選擇性的萃取水溶液中的胺基酸硫醇類小分子並結合毛細管電泳吸收儀進行偵測
一、前言1
二、藥品與方法5
2.1、藥品5
2.2、檢測金奈米粒子之儀器設備7
2.3、合成金奈米粒子7
2.4、萃取過程(Extraction procedure)8
2.5、毛細管電泳偵測系統10
2.6、分析尿液中的Aminothiols與肌酸酐(Creatinine)11
三、結果與討論12
3.1、利用Tween 20-AuNPs選擇性萃取Aminothiols12
3.2、AuNPs 和 DTT 的濃度變化及樣品的體積變化對於水溶液中Aminothiols萃取濃縮效率之影響16
3.3、Aminothiols定量與真實樣品之應用22
四、結論28
五、參考文獻29

第二章、利用檸檬酸鈉金奈米粒子萃取吲哚胺類並結合毛細管電泳雷射誘導自發螢光法進行偵測
一、前言36
二、藥品與方法40
2.1、藥品40
2.2、合成金奈米粒子(Citrate-AuNPs)41
2.3、金奈米粒子(Citrate-AuNPs)的特性42
2.4、毛細管電泳(Capillary Electrophoresis)偵測系統43
2.5、萃取過程(Extraction procedure)44
2.6、分析尿液中的吲哚胺類(Indoleamines)46
三、結果與討論48
3.1、分離吲哚胺類(Indoleamines)的最佳化條件48
3.2、利用檸檬酸鈉金奈米粒子(Citrate-AuNPs)對吲哚胺類 (Indoleamines)進行萃取 50
3.3、靈敏度與定量57
3.4、分析尿液中的吲哚胺類(Indoleamines )62
四、結論66
五、參考文獻 67
第一章、利用非離子型界面活性劑修飾之金奈米粒子選擇性的萃取水溶液中的胺基酸硫醇類小分子並結合毛細管電泳吸收儀進行偵測
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