臺灣博碩士論文加值系統

本論文主要分成兩個部分，在第一部份中，利用調控兩種不同粒徑 (平均直徑：3.5和14 nm) 的金奈米粒子之混合比例當做選擇性探針，同時做為表面輔助雷射脫附游離質譜儀 (surface-assisted laser desorption/ionization mass spectrometry ，SALDI-MS) 的基質，以期待能選擇性偵測生物樣品中的胺基酸硫醇 (aminothiol)。由於3.5 nm的金奈米粒子相較於粒徑更大的14 nm奈米粒子而言，對於胺基硫醇有較好的脫附與游離效率；但是若要以離心的方式來濃縮粒徑小的3.5 nm金奈米粒子卻又有一定的困難度。為了解決這個問題，我們利用3.5和14 nm的金奈米粒子混合溶液，藉由胺基酸硫醇的存在下會使金奈米粒子聚集而較易離心，使得濃縮的效果提升。當3.5和14 nm的金奈米粒子的濃度分別為38和150 pM時，選擇性偵測溶液 (1.0 mL) 當中的谷胱甘肽 (glutathione，GSH)、半胱胺酸 (cysteine，Cys)和同半胱胺酸 (homocysteine，HCys) 時，可以得到偵測極限分別為2、20和44 nM (S/N＝3)。由於本方法有很好的靈敏度、再現性及樣品處理簡單的優點，此方法已成功證明可以應用於分析MCF-7細胞株及血漿中的GSH和Cys的含量。
在第二部份中，主要是合成並探討奈米材料，包括金奈米粒子(Au NPs)、鉑奈米海綿 (Pt NSPs)、磁性四氧化三鐵奈米粒子 (Fe3O4 NPs)、二氧化鈦奈米粒子 (TiO2 NPs)、硒奈米粒子 (Se NPs) 以及銻化鎘量子點 (CdTe QDs) 等六種奈米粒子，應用於SALDI-MS中分析胺基酸硫醇，胜肽及蛋白質分子之結果。由於選用奈米粒子當做基質時雖然可以成功的偵測生物分子，但是SALDI-MS的靈敏度、再現性及分析的質量上限等，與奈米粒子的特性和濃度有極大的關係，本研究中主要希望能夠對實驗室開發的無機材料奈米粒子，適合於何種類型或適合何種分子量的生化分子，以及其分析的條件(包括奈米粒子濃度、雷射強度及緩衝溶液濃度等) 做一個總括的探討。實驗結果發現，金奈米粒子對於偵測小分子 (GSH) 有最低的偵測極限 (140 fmol)；而利用Pt NSPs 及Fe3O4 NPs當作SALDI-MS基質時，可測到最高的質量上限 (25 kDa)。此原因可能是分析物在雷射光照射下進行有效之脫附游離，並且分析物與奈米材料間作用力不應過強而使分析物不易脫附。此外，在雷射光照射下奈米材料也應處穩定，不易產生金屬團簇訊號而降低分析物偵測靈敏度。若選用Fe3O4 NPs和CdTe QDs當做基質時，質譜圖譜中也會發現有Fe3+和Cd2+加成的訊號產生，雖然此種方式能夠增加分析物於SALDI-MS脫附游離的效率，但也使其訊號變寬而降低圖譜解析度。

This thesis is divided into two parts. In the first part, we have employed two sets of differently sized (average diameters: 3.5 and 14 nm) gold nanoparticles (Au NPs) as selective probes and matrices for the determination of aminothiols in biological samples using surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). Because of their higher ionization efficiency, the 3.5-nm-diameter Au NPs exhibited greater desorption/ionization efficiency toward the aminothiols than did the larger Au NPs; centrifugation of these small Au NPs was, however, difficult. To solve this problem, we investigated the use of mixtures of the 3.5- and 14-nm Au NPs; in this case, the analyte-induced Au NP aggregates were readily centrifuged, providing greater concentration efficiency. When using 38 and 150 pM solutions of the 3.5- and 14-nm Au NPs, respectively, as the probe and matrix, SALDI-MS provided limits of detection (signal-to-noise ratio = 3) of 2, 20, and 44 nM for 1.0-mL solutions of glutathione (GSH), cysteine (Cys), and homocysteine, respectively. We validated the practicality of this approach—with its advantages of sensitivity, reproducibility, rapidity, and simplicity—through the analysis of GSH in MCF-7 cell lysates and Cys in plasma.
In the second part, we have synthesized and investigated the uses of nanomaterials, including Au NPs, Pt nanosponges (NSP), Fe3O4 NPs, TiO2 NPs, Se NPs, and CdTe quantum dots (QDs), as SALDI-MS matrixes for the analysis of small molecules, peptides and proteins. Although those nanomaterials served as useful inorganic matrixes in the SALDI-MS measurements, SALDI-MS performance with respect to sensitivity, reproducibility, and mass range is highly dependent on the nature and concentrations of NPs. Thus, our aim of this study is to evaluate different NP matrixes for analyzing biomolecules in those optimum conditions. We found that for small solutes (e.g. glutathione), Au NPs provided the lowest (140 fmol) limit of detection (LOD) at signal-to-noise ration 3. And the upper detectable mass range is approximately 25 kDa by using Pt NSPs and Fe3O4 NPs as the SALDI-MS matrix. For efficient desorption and ionization, nanomaterials need to have a strong absorption of laser energy and an efficient proton energy transfer to the analytes, while their interactions with analytes can not be too strong. With respect to sensitivity, the nanomaterials must be stable under laser irradiation; minimum formation of clusters from the nanomaterials such as Au clusters. Although Fe3+ and Cd2+ have strong interactions with proteins, leading to improved ionization efficiency (sensitivity), they cause peak broadening (loss in mass resolution).

目錄
中文摘要I
英文摘要III
目錄V
表目錄IX
圖目錄X

第一章 緒論
1.1前言1
1.2基質輔助雷射脫附游離質譜法 (MALDI-MS)2
1.2.1MALDI-MS的發展歷史2
1.2.2MALDI-MS樣品製備3
1.2.3有機基質的特性與功能4
1.2.4樣品脫附游離的機制6
1.2.5飛行時間 (Time-of-Flight，TOF) 質量分析器13
1.2.6影響分析物訊號強弱的因素15
1.3表面輔助雷射脫附游離 (SALDI-MS) 質譜法16
1.3.1SALDI-MS的發展歷史16
1.3.2SALDI-MS樣品脫附游離的機制17
1.3.3SALDI-MS基質的應用18
1.3.3.1金奈米粒子18
1.3.3.2氧化鐵奈米粒子19
1.3.3.3二氧化鈦奈米粒子20
1.3.3.4奈米碳管21
1.3.3.5矽奈米材料22
1.4研究動機23
1.4.1利用尺寸不同的金奈米粒子選擇性偵測胺基酸硫醇23
1.4.2不同奈米粒子作為SALDI-MS之基質於胜肽及蛋白質分析25
1.5參考文獻25

第二章 利用尺寸不同的金奈米粒子選擇性偵測胺基酸硫醇
2.1前言29
2.2實驗部分31
2.2.1實驗藥品31
2.2.2合成金奈米粒子31
2.2.3金奈米粒子之光譜鑑定33
2.2.4標準樣品的製備34
2.2.5實樣品的處理35
2.2.6MALDI-TOF-MS36
2.3結果與討論真36
2.3.1以金奈米粒子作為SALDI-MS基質36
2.3.2以金奈米粒子作為選擇性偵測之探針38
2.3.3pH值和鹽類的影響42
2.3.4不同胺基酸硫醇的分析45
2.3.5對三種胺基酸硫醇的定量47
2.3.6分析乳癌細胞 (MCF-7) 中胺基酸硫醇的含量51
2.3.7分析血漿中胺基酸硫醇的含量52
2.4結論53
2.5參考文獻53

第三章 不同奈米粒子作為SALDI-MS之基質於胜肽及蛋白質分析
3.1前言56
3.2實驗部分58
3.2.1實驗藥品58
3.2.2合成金奈米粒子59
3.2.3合成鉑奈米海綿59
3.2.4合成磁性四氧化三鐵奈米粒子60
3.2.5合成二氧化鈦奈米粒子60
3.2.6合成硒奈米粒子60
3.2.7合成碲化鎘量子點60
3.2.8奈米粒子之鑑定61
3.2.9樣品的製備68
3.2.10MALDI- TOF-MS69
3.3結果與討論69
3.3.1六種奈米粒子作為SALDI-MS基質的結果69
3.3.2熱傳導效率及形狀對分析的影響75
3.3.3緩衝溶液對分析的影響78
3.3.4分析的質量上限78
3.3.5定量分析80
3.4結論81
3.5參考文獻81

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