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研究生:葉佩蓉
研究生(外文):Pei-Rong Yeh
論文名稱:金奈米粒子萃取劑結合毛細管電泳分析溶菌酶
論文名稱(外文):Gold nanoparticle extraction combined with capillary electrophoresis for analyzing lyzoyme
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:2012
畢業學年度:100
語文別:中文
論文頁數:82
中文關鍵詞:高等電點蛋白質溶菌酶奈米粒子萃取毛細管電泳金奈米粒子
外文關鍵詞:lysozymehigh-pI proteinsnanoparticle extractioncapillary electrophoresisgold nanoparticle
相關次數:
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本篇研究使用人類血清白蛋白修飾的金奈米粒子(human serum albumin-capped gold nanoparticles,HSA-AuNPs)對溶菌酶(Lysozyme,Lyz)進行選擇性濃縮萃取,並結合毛細管電泳進行偵測。因HSA-AuNPs在高鹽類環境下具良好的穩定性,且在中性的萃取條件下(pH=7.0),HSA與Lyz間具有很強的靜電作用力,故HSA-AuNPs可自一複雜的基質中萃取出Lyz。藉由在背景電解質中添加1.2%陽離子聚合物-聚二烯丙基二甲基氯化銨(Poly(diallyldimethylammonium chloride),PDDAC),能有效避免蛋白質吸附於毛細管壁,進而成功偵測到Lyz。當萃取步驟結束後,不須使用釋放試劑,即可直接進行毛細管電泳分析,與一般的奈米粒子萃取方法相比,不僅可省去釋放試劑的使用,也可縮短整個前處理步驟所需的時間。由於Lyz的萃取效率取決於環境的pH值、HSA-AuNPs的濃度以及與HSA-AuNPs的反應時間,故對這三者因素進行最佳化條件的探討。在最佳化條件下,對Lyz進行定量分析,由3倍的訊號與雜訊比(S/N=3),得到Lyz的偵測極限為8 nM,而線性範圍為20~400 nM。本篇透過結合HSA-AuNPs的萃取與充滿PDDAC的毛細管電泳分離技術下,能成功應用於分析雞蛋蛋白、人類眼淚及人乳中的Lyz。除此之外,本篇所開發之萃取技術也可結合基質輔助雷射脫附/游離質譜技術(MALDI)與十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(SDS-PAGE)進行分析。
This study describes the use of human serum albumin (HSA)-modified gold nanoparticles (HSA-AuNPs) for the selective extraction and enrichment of high-pI protein, lysozyme (Lyz) prior to analysis by capillary electrophoresis (CE) with UV detection. HSA-AuNPs are capable of extracting Lyz from a complicated matrix because a HSA capping layer not only stabilizes gold nanoparticles in a high-salt environment but also exhibits strong electrostatic attraction with Lyz under neutral pH condition. Efficient separation of Lyz and other high-pI proteins has been successfully achieved by the filling of cationic polyelectrolyte, poly(diallydimethylammonium chloride) (PDDAC), to the background electrolyte. After capturing Lyz with HSA-AuNPs, PDDAC-filled CE can be directly used for the analysis of the extracted Lyz without the addition of the releasing agent into the extractor. The extraction efficiency relied on the pH of the solution and the concentration of HSA-AuNPs. Under optimal extraction conditions, the limits of detection at a signal-to-noise ratio of 3 for Lyz were down to 8 nM. The combination of HSA-AuNP extraction and PDDAC-filled CE has been applied the analyses of lysozyme in chicken egg white, white wine and human tear. Also, we reveal that this NP-based extraction can be coupled to matrix-assisted desorption/ionization time-of-flight mass spectrometry.
謝誌 ⅰ
摘要 ⅱ
Abstact ⅲ
目錄 ⅳ
圖表目錄 ⅵ
縮寫表 ⅷ
第一章、緒論 1
一、毛細管電泳(capillary electrophoresis,CE)簡介 1
1.1 CE的發展歷史 1
1.2 CE的分離原理 1
1.3 CE的進樣方式 8
1.4 CE的偵測方式 10
1.5毛細管電泳管壁塗覆方法 11
二、奈米材料的簡介 14
2.1 奈米材料的性質 14
2.2 奈米材料的製備 15
2.3 金奈米粒子的簡介 21
2.4 金奈米粒子在生醫上的應用 22
三、研究動機與目的 24
第二章、實驗部分 26
一、藥品與溶液製備 26
二、合成修飾人類血清白蛋白的金奈米粒子(HSA-AuNPs) 28
1. 合成Citrate-AuNPs 28
2. 合成修飾人類血清白蛋白的金奈米粒子(HSA-AuNPs) 28
三、儀器裝置 29
四、萃取過程 33
五、真實樣品 33
第三章、結果與討論 35
一、證實HSA-AuNPs濃縮萃取效果 35
二、探討HSA-AuNPs與Lyz之間的作用力 38
三、萃取參數的最佳化條件 44
四、選擇性與靈敏度 48
五、再現性探討 52
六、應用於MALDI-TOF MS 與SDS-PAGE 53
七、真實樣品分析 56
第四章、結論 61
第五章、參考資料 62


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