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研究生:劉映君
研究生(外文):Yin-Chun Liu
論文名稱:利用雷射脫附游離質譜探討金奈米粒子表面特性
論文名稱(外文):Investigation of the Surface of Gold Nanoparticles by Laser Desorption/Ionization Mass Spectrometry
指導教授:黃志清黃志清引用關係
指導教授(外文):Chih-Ching Huang
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
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:85
中文關鍵詞:表面輔助雷射脫附游離鉛離子凝血酶生物樣品金團簇
外文關鍵詞:SALDIlead ionthrombinbiofluidgold cluster
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本論文分為兩部分。主要是利用金奈米粒子 (Au NPs)作為基質 (matrix),以雷射脫附游離質譜法 (laser desorption/ionization mass spectrometry, LDI-MS)分析金屬離子的團簇 (cluster)訊號可分別偵測鉛離子 (lead ion, Pb2+)及凝血酶 (thrombin)。第一部份是將修飾牛血清白蛋白 (bovine serum albumin, BSA)的Au NPs (BSA–Au NPs) 藉由疏水性作用力吸附於硝基纖維膜 (nitrocellulose membrane, NCM)上製成LDI-MS基質 (BSA–Au NP/NCM)後,可偵測並定量複雜生物樣品 (尿液和血液)中的Pb2+至nM等級。當溶液中存在硫代硫酸根 (thiosulfate, S2O32-)時,會與Au NPs形成Au+•S2O32–錯合離子,並加速Pb2+沈積在Au NPs表面。在最佳化條件下,BSA–Au NP/NCM基質於LDI-MS中對Pb2+的偵測靈敏度高 (limit of detection, LOD = 0.5 nM),選擇性高於其他金屬離子100倍。第二部分是將29個鹼基對組成的凝血酶適合體 (Thrombin-binding aptmaer, TBA29)修飾於Au NPs (TBA29–Au NPs),與凝血酶反應並經簡單的純化步驟後,吸附於NCM上製成TBA29–Au NP/NCM基質。由於TBA29與凝血酶專一性鍵結,使得雷射能量無法順利傳遞至Au NPs使之脫附游離,造成金團簇訊號隨著凝血酶濃度上升而逐漸降低,透過金團簇訊號的下降率,可於血清環境偵測凝血酶至fM。在最佳化條件下,TBA29–Au NP/NCM對凝血酶的偵測具有高靈敏度 (LOD ~50 fM)和選擇性 (為其他蛋白質的1000倍)。
We have developed the mass spectrometric detection of lead ions (Pb2+)and thrombin through the analysis of metal clusters from gold nanoparticles (Au NPs) via laser desorption/ionization (LDI). A matrix-free strategy that based on the analysis Au NP–modified nitrocellulose membranes (NCMs) using LDI-mass spectrometry (LDI-MS) was used to comprehensive quantification of lead ions (Pb2+), with subnanomolar sensitivity, in complicated biofluids. Strong hydrophobic interactions between the NCM and bovine serum albumin (BSA) led to trapping of BSA–modified Au NPs (BSA–Au NPs), resulting in the formation of a nanocomposite film of BSA–Au NPs on the membrane (BSA–Au NP/NCM). When the Au NPs interacted with thiosulfate (S2O32–) ions in solution, Au+•S2O32– complexes formed on the Au NP surfaces, facilitating the deposition of Pb atoms in the form of PbAu alloys in the presence of Pb2+ ions. Using the BSA–Au NP/NCM substrate as an LDI-MS matrix allowed the soft and enhanced ionization of Pb–Au alloys from the Au NP surfaces to obtain accurate mass measurement (precision: 5 ppm) of Au, Pb, and Au–Pb species. Under optimal conditions, this LDI-MS approach provided high sensitivity (limit of detection, LOD = 0.5 nM) and selectivity (by at least 100-fold in terms of tolerance concentrations) toward Pb2+ ions. Moreover, we unveil a new LDI-MS approach to the detection of proteins with femtomolar sensitivity through the analysis of gold (Au) clusters from aptamer–modified gold nanoparticles (Apt–Au NPs) on a NCM. As the target protein is selectively captured by surface aptamers, those particles were isolated by a simple centrifugation process and then deposited on the NCM to form a highly efficient background-free LDI substrate. On the basis of the principle of target protein-bound Apt–Au NPs inhibit the efficient energy absorption and/or transfer of surface Au atoms that giving rise to a low abundance of Au cluster ions in the mass spectrum, the decrease in the signal intensities of Au cluster ions as a target-labeling indicator of target proteins. Under optimal conditions, this probe is highly sensitive (limit of detection ~50 fM) and selective (by at least 1000-fold over other proteins) toward thrombin.
謝辭 i
中文摘要 ii
Abstract iv
第一章 緒論 1
1.1 表面輔助雷射脫附游離質譜法 (Surface-assisted laser desorption/ionization mass spectrometry, SALDI-MS) 1
1.1.1 SALDI-MS的發展歷史 1
1.1.2 SALDI-MS樣品脫附游離的機制 2
1.1.3 金奈米粒子於SALDI-MS基質的應用 3
1.1.3.1 未修飾之金奈米粒子應用 4
1.1.3.2 修飾之金奈米粒子應用 6
1.1.3.3 其他形式之應用 7
1.2 研究目的 9
1.4 參考文獻 10
第二章 利用功能性金奈米薄膜結合雷射脫附游離質譜儀偵測生物樣品中之鉛離子 12
2.1 前言 12
2.2 實驗材料與方法 13
2.2.1 實驗藥品 13
2.2.2 儀器設備 14
2.2.3 金奈米粒子的合成 15
2.2.4 Glycine-NaOH緩衝溶液配製 16
2.2.5 製備BSA–Au NP/NCM基質 16
2.2.6 以BSA–Au NP/NCM檢測鉛離子 17
2.2.7 尿液樣品之檢測 18
2.2.8 全血樣品之檢測 18
2.2.9 檢測血液標準品中的鉛離子 19
2.3 結果與討論 19
2.3.1 製備BSA–Au NP/NCM基質 19
2.3.2 以BSA–Au NP/NCM與LDI-MS偵測鉛離子 20
2.3.3 探討BSA–Au NP/NCMs於LDI-MS中之最佳化條件 22
2.3.4 金屬選擇性 24
2.3.5 偵測Pb2+之靈敏度 25
2.3.6 全血及尿液樣品中之鉛離子分析 25
2.4 結論 26
2.5 參考文獻 28
2.6 本章圖表 31
第三章 藉由分析質譜圖中金奈米粒子之團簇訊號偵測蛋白質 44
3.1 前言 44
3.2 實驗材料與方法 45
3.2.1 實驗藥品 45
3.2.2 儀器設備 46
3.2.3 金奈米粒子的合成 46
3.2.4 仿生理緩衝溶液 (Physiological buffer solution, PBS)配製 47
3.2.5 合成TBA29–Au NPs 47
3.2.6 利用TBA29–Au NP/NCM基質與LDI-MS偵測凝血酶 48
3.2.7 於血清環境中偵測凝血酶 48
3.3 結果與討論 49
3.3.1 以TBA29–Au NPs結合LDI-MS偵測凝血酶 49
3.3.3. 探討TBA29–Au NP/NCMs於LDI-MS中之最佳化條件 52
3.3.4. 對凝血酶的選擇性及靈敏度探討 52
3.3.5. 於血清環境下偵測凝血酶 53
3.4 結論 54
3.5 參考文獻 56
3.6 本章圖表 63

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