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研究生:陳慎泓
研究生(外文):Shen-Hong Chen
論文名稱:金屬螯合壓電晶體生物感測晶片之研製
論文名稱(外文):Preparation of metal-chelating piezoelectric biosensor
指導教授:陳秀美陳秀美引用關係
指導教授(外文):Hsiu-Mei Chen
學位類別:碩士
校院名稱:國立臺灣科技大學
系所名稱:化學工程系
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:1999
畢業學年度:87
語文別:中文
論文頁數:135
中文關鍵詞:壓電晶體固定化金屬親和層析法金屬親和標籤日本血吸蟲穀胱甘呔轉移酵素金屬螯合
外文關鍵詞:piezoelectric crystalImmobilized metal-affinity chromatographyIMACaffinity tagGlutathione — S — transferaseGSTmetal chelating
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  • 被引用被引用:7
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固定化金屬親和層析法(Immobilized metal-affinity chromatography,IMAC)為利用固定有金屬離子的載體而將一具有金屬親和性的蛋白質與其他蛋白質分離的一種生物分離技術,普遍應用於基因重組蛋白之純化上。本研究便應用此原理,製備一可螯合Ni2+離子的壓電晶體生物感測晶片,檢測帶有金屬親和標籤(Poly - His tag)的日本血吸蟲穀胱甘呔轉移酵素(SjGST/His),並應用於其抗體之檢測。在晶片製備上,以所合成的十五烷基酸硫醇(15-mercaptopentadecanoic acid,15 - MA)修飾壓電晶體金面,並固定化金屬螯合劑(nitrilotriacetic acid,NTA)以螯合Ni2+離子。研究結果顯示:15 - MA經DSC、FTIR、及GC-Mass之鑑定分析後,可確定其成份之正確性。其於晶體金面上之吸附修飾約需3天才達到飽和,且在pH值為6時,NTA固定到晶片上的量最高。此外經由ESCA分析可證實晶片上15 - MA及NTA的存在,且可螯合Ni2+離子;而由AFM的表面分析,可看出晶片在不同修飾階段的表面性狀差異性。在金屬離子之螯合分析上,固定有NTA的壓電晶體依據其在乾燥狀態下所測得之頻率值,可知其對Ni2+之最低檢測濃度可達10-15 M。在靜止液相下於10-3∼10-5 M的Ni2+離子濃度內,其頻率應答呈線性增加關係。而在注流式分析下對於Ni2+離子之吸附,修飾有NTA晶片的頻率應答值較沒有NTA修飾晶片的應答值為高;。最後在SjGST/His之檢測上,在乾燥狀態下測得有22917 Hz的非特異性吸附值。在注流式分析下螯合有Ni2+離子的壓電晶片比沒有螯合離子之晶片可較快速地檢測到SjGST/His,且應答值較高。其線性檢測範圍為0.1  g/ml~40 g/ml蛋白質濃度內,且可在進一步地檢測其抗體,但是仍必須克服非特異性吸附之問題。
Immobilized metal-affinity chromatography, IMAC, is a well known bioseparation technique, which uses metal-chelating adsorbent to selectively purify metal-affinity proteins from contaminants. In this study, IMAC was applied to prepare a metal-chelating piezoelectric(PZ)biosensor for the detection of SjGST/His carrying a metal-affinity tag, poly-His tag, and for the subsequent detection of its antibody. To prepare the biosensor, synthesized 15-mercaptopentadecanoic acid, 15-MA, was first used to fabricate the gold electrode of the PZ crystal as a self-assembled monolayer, and a metal-chelating ligand, nitrilotriacetic acid, NTA, was then covalently immobilized.The accuracy of the synthesized 15-MA was confirmed by DSC, FTIR, and GC-Mass analysis. Three days were required for the fabrication of 15-MA onto the gold electrode, and the optimal pH for NTA immobilization was 6. The analysis by using ESCA further confirmed the existence of 15-MA and NTA on the surface of the prepared biosensor, as well as the chelated Ni2+ ion. In addition, the morphology of the crystal surface at each modification step was examined by using AFM. For metal-chelating, the lowest Ni2+ concentration detected by the prepared biosensor was 10-15 M, according to the frequency shift value after drying, while the liner detection range of the crystal in a flow-injection analysis system was from 10-3 to 10-5 M Ni2+ concentrations. For the detection of SjGST/His, highly non-specific binding of SjGST/His onto the NTA modified surface was observed under either a non-flow liquid detection or a flow-injection analysis system. However, the biosensor chelated with Ni2+ detected SjGST/His at a higher and a faster response rate than did the biosensor without Ni-chelating, and a liner detection range of 0.1 to 40 g/ml protein concentration was obtained. Finally, a preliminary study suggested that the detection of anti-SjGST antibody by the SjGST/His captured by Ni2+ chelated on the prepared PZ biosensor is feasible.
中文摘要…………………………………………………….………..I
英文摘要……………………………………………………………..II
誌 謝……………………………………………………………….III
目 錄……………………………………………………………….VII
表目錄VIII
圖目錄IX
第一章 緒論1
第二章 文獻回顧4
2-1生物感測器4
2-1-1 生物感測器及其分類4
2-2-1 生物感測器的應用9
2-2壓電晶體生物感測器10
2-2-1 壓電晶體10
2-2-2 壓電晶體感測原理11
2-2-3 壓電晶體生物感測器的研究發展14
2-3生物感測晶片的表面修飾15
2-3-1 生物感測晶片之活化修飾:16
2-3-2 生物感測晶片之蛋白質固定化20
2-4固定化金屬親和層析(IMAC)29
2-4-1 IMAC原理29
2-4-2 蛋白質之金屬螯合機制32
2-4-3 螯合劑35
2-4-4 IMAC之應用37
2-5榖胱甘呔轉移酵素(SjGST/His)39
2-6材料表面特性之分析方法41
2-6-1 表面幾何形狀分析42
2-6-2 化學分析電子分光儀(ESCA)44
2-7 研究目的45
第三章 實驗47
3-1實驗設備與方法47
3-1-1 實驗設備47
3-1-2 實驗藥品51
3-1-3 藥品配製52
3-2實驗流程54
3-3實驗步驟57
3-3-1 十五烷基酸硫醇(15-MA)之合成與鑑定57
3-3-2 壓電晶體表面定性分析57
3-3-3 可螯合金屬離子之壓電晶體感測晶片的製備58
3-3-4 金屬離子之螯合分析59
3-3-5 SjGST/His的檢測60
第四章 結果與討論63
4-1石英壓電晶體微量天平之校正63
4-2十五烷基酸硫醇之鑑定65
4-2-1 DSC之鑑定65
4-2-2 GC-Mass67
4-2-3 FTIR72
4-3十五烷基酸硫醇之表面修飾74
4-4 NTA於壓電晶體晶體之固定化74
4-5材料表面之定性分析78
4-5-1 表面元素分析 78
4-5-2 AFM表面性質分析82
4-6金屬離子之螯合分析87
4-6-1 於乾燥狀態下87
4-6-2 於靜止液相系統中87
4-6-3 於FIA系統分析中88
4-7 SjGST/His之檢測95
4-7-1 於乾燥狀態下95
4-7-2 於靜止液相系統中96
4-7-3 於FIA系統分析中97
第五章 結論109
參考文獻112
附錄122
A. 固定有NTA螯合劑之親水性壓電晶體之製備122
A-1 15-MA/POD/NTA之製備法122
A-2 15-MA/Dextran/NTA之製備法124
B. 固定有NTA螯合劑之壓電晶體之表面性質分析133
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