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研究生:王安妮
研究生(外文):Anne Wang
論文名稱:奈米複合材料用於蛋白質體分析之研究
論文名稱(外文):Nanocomposite materials for Proteomics Analysis
指導教授:陳淑慧陳淑慧引用關係
指導教授(外文):Shu-Hui Chen
學位類別:碩士
校院名稱:國立成功大學
系所名稱:微機電系統工程研究所
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:99
中文關鍵詞:蛋白質體高分子金奈米粒子
外文關鍵詞:PDMSproteomicsgold nanoparticle
相關次數:
  • 被引用被引用:1
  • 點閱點閱:158
  • 評分評分:
  • 下載下載:17
  • 收藏至我的研究室書目清單書目收藏:1
在蛋白質體研究中,樣品的前處理包括樣品的濃縮、純化和去鹽以減少樣品的複雜性是目前在接下來質譜的偵測上很重要的實驗關鍵。 奈米粒子本身因為具有高的表比面積,且和蛋白質尺寸是相近,近年來奈米粒子在蛋白質體分析的樣品前處理中引起廣大的興趣。因此,在本研究中,我們針對金奈米粒子發展其在蛋白質的前處理之濃縮及親合性鍵結上的應用技術。
在第一部份的研究中,針對易取得的大量尿液樣品(含少量蛋白),我們使用金奈米粒子可以容易地從大體積(15 mL)中濃縮蛋白(最後濃度1.4 ppm),進一步進行一維膠電泳可將金奈米粒子和蛋白質的沉澱物分開並使蛋白質能夠在一為膠電泳中分離,之後進行膠體內消化,以串聯式質譜儀(LC/MS/MS)做偵測,結果顯示金奈米粒子不僅是一個簡單的蛋白濃縮方法且適用於大體積低濃度的生物樣品
在第二部份的研究中,我們發展以金奈米粒子修飾的PDMS為基材之蛋白質晶片,利用多電層堆疊的方式修飾PDMS表面,且此蛋白質晶片也設計於可用螢光或質譜做偵測。在多電層的修飾中,我們使用高分子PEI和PAA一層一層的堆疊在PDMS表面上,經過這樣的修飾後,長久性親水性的PDMS表面形成且其表面存有活化官能基(胺機或基)能進一步的衍生化。最上層為PEI的表面,我們舖上一層密度高且均勻的金奈米粒子,接著,固定衍生化的雌激素(E2 -NH2)於此表面上,並用雌激素接受器(ER α)做親合性鍵結的測試,由質譜儀(MALDI-TOF/MS) 偵測;而生物素(biotin)則利用醯胺鍵結在PAA的表面上,由螢光標記的卵白素(streptavidin)做螢光測試,由這些結果顯示我們修飾的PDMS之蛋白質晶片可應用於親合性生物分子的檢測。
Sample preparation steps such as concentration, purification, and desalting to reduce the complexity prior to MS analysis are essential but challenging for proteomic research. Recently, nanoparticles have gained great interests in sample preparation for proteomic analysis because of their large surface to volume ratio and comparable size with protein molecules. In this study, we focused on developing efficient sample cleanup and affinity capturing techniques using Au nanoparticles(Au-NP) for MS-based proteomic analysis.
In the first part, Au-NP was applied to concentrate proteins from more than 15 mL of human urine, in which the total protein concentration is less than 1.4 ppm. Moreover, Au-NP-aggregated proteins could be directly applied to gel electrophoresis for protein dissociation from Au-NP aggregates, and then followed by free protein separation as well as for the subsequent in-gel digestion and protein identification by mass spectrometry. Results indicate that Au-NP aggregation is not only a simple and efficient method for enriching a broad range of proteins, and it is particularly useful for concentrating proteins from a relatively large volume of dilute biological fluids.
In the second part, we developed a Au-NP modified PDMS substrate for protein chips using layer-by-layer techniques. The developed protein chips are designed to be comparable for both MS and fluorescence detection. In this modification, PEI and PAA polymer was coated in sequence. After such modification, a long term hydrophilic surface could be formed on the PDMS surface and the functional group, amine or carboxylic acid group, was able to be derivative for the following steps. To the top PEI layer, a dense and homogenous Au-NP layer was formed by electrostatic force and amine-derivatized estrogen molecules were immobilized and identified by applying estrogen receptor proteins using MALDI/MS detection. Biotin molecule was immobilized on the top PAA layer through the reduction amination and identified by applying streptavidin-cy3 using fluorescence detection. These results demonstrate that the modified PDMS substrate is capable for sensing biomolecular interactions.
目錄
中文摘要-------------------------------------------------------------------I
英文摘要------------------------------------------------------------------ III
致謝-----------------------------------------------------------------------IV
發表期刊-------------------------------------------------------------------VI
主文目錄-------------------------------------------------------------------XI
表目錄-------------------------------------------------------------------- XVII
圖目錄--------------------------------------------------------------------XVIII

第一章 序論
1.1 奈米時代------------------------------------------------------------1
1.2 蛋白質體的發展------------------------------------------------------2
1.3奈米技術和蛋白質體的結合---------------------------------------------3

第二章 金奈米粒子在蛋白質體的應用
2.1尿液概論-------------------------------------------------------------5
2.1.1尿液形成和組成---------------------------------------------------5
2.1.2尿液的分析-------------------------------------------------------6
2.2蛋白濃縮方法---------------------------------------------------------7
2.3金奈米粒子-----------------------------------------------------------8
2.4蛋白質體學方法在尿液研究上的應用-------------------------------------8
2.4.1電噴灑離子化-液相層析串聯質譜儀------------------------------------8
2.4.2一維膠電泳: SDS 聚丙烯醯胺電泳-------------------------------------9
2.4.3尿液之蛋白質體學分析的文獻回顧及研究方向--------------------------10
2.5實驗部分
2.5.1實驗藥品及儀器----------------------------------------------------12
2.5.2實驗方法
2.5.2.1蛋白質的變性----------------------------------------------------17
2.5.2.2金奈米粒子的製備--------------------------------------------17
2.5.2.3蛋白質的濃縮------------------------------------------------17
2.5.2.4膠體的製備--------------------------------------------------18
2.5.2.5樣品的分離--------------------------------------------------19
2.5.2.6膠體的染色--------------------------------------------------19
2.5.2.7膠體內的消化------------------------------------------------20
2.5.2.8質譜的分析--------------------------------------------------21
2.6結果與討論
2.6.1 UV和TEM的鑑定--------------------------------------------------22
2.6.2標準蛋白質的濃縮------------------------------------------------22
2.6.3尿液的濃縮及鑑定------------------------------------------------25
2.7結論----------------------------------------------------------------28

第三章 高分子材質在蛋白質親合性反應的應用
3.1蛋白質晶片的發展----------------------------------------------------44
3.2聚雙甲基矽氧烷( poly(dimehtylsiloxane), PDMS)-----------------------45
3.3 雌激素(Estrogens)與雌激素接受器(Estrogen Receptor)-----------------46
3.4 PDMS為基材之蛋白質晶片及其表面化學---------------------------------47
3.4.1實驗藥品與儀器--------------------------------------------------48
3.4.2實驗方法
3.4.2.1 Polyelectrolyte multilayer on MALDI plate------------------55
3.4.2.2表面分析----------------------------------------------------56
3.4.2.3螢光測試----------------------------------------------------57
3.4.2.4 PDMS上金奈米粒子的固定-------------------------------------57
3.4.2.5生物分子固定在金奈米粒子表面--------------------------------58
3.4.2.6晶片上的酵素消化--------------------------------------------58
3.4.2.7基質輔助雷測脫附離子源-飛行時間質譜儀-----------------------58
3.4.2.8 Hemoglobin-ELISA的螢光和質譜測試---------------------------59
3.5結果與討論
3.5.1 Polyelectrolyte multilayer的鑑定-------------------------------60
3.5.2螢光測試生物分子之固定化----------------------------------------61
3.5.3衍生化雌激素E2的鑑定----------------------------------------------63
3.5.4 PDMS表面金奈米粒子的鑑定---------------------------------------64
3.5.5利用質譜儀測試衍生化雌激素(E2-NH2)之固定化----------------------64
3.5.6雌激素受器的質譜鑑定--------------------------------------------65
3.5.7 Hemoglobin-ELISA的螢光和質譜鑑定---------------------------------66
3.6結論----------------------------------------------------------------68
第四章 總結與未來展望--------------------------------------------------95
参考文獻---------------------------------------------------------------96
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