臺灣博碩士論文加值系統

金奈米粒子具體積小、比表面積大、且表面容易進行化學修飾等特性，而以檸檬酸鈉還原四氯金酸所製備出的金奈米粒子，因為表面吸附有檸檬酸根離子和其他反應後的陰離子而使金奈米粒子表面具有負電性，本論文利用此特性發展一奈米探針，可以用來選擇吸附帶正電荷的分析物，為了使金奈米粒子在萃取濃縮分析物後的分離更簡便，本論文嘗試將金奈米粒子修飾在磁性粒子的表面，發展成表面帶有負電荷的Fe-Au探針，利用磁鐵可以將這些完成萃取的
Fe-Au探針很容易的從樣品溶液中分離出來，而達到快速分離的效果，並結合基質輔助雷射脫附游離質譜法 (MALDI-MS) 做為確認分析的方法。

本論文已成功地將此探針應用於濃縮水溶液樣品中微量的陽離子型界面活性劑、胜肽及蛋白質等等；並且也可藉著改變樣品溶液中pH值，可以選擇抓取樣品中特定的胜肽或蛋白質分子。另外針對蛋白質消化後的胜肽片段樣品
，由於其濃度通常不高，直接使用MALDI-MS不容易偵測到胜肽片段的訊號，但只要將溶液的酸鹼值調整至pH值為4左右，即可使大部分的胜肽片段帶上淨正電荷因而被Fe-Au探針順利抓取。實驗結果顯示細胞色素c經過胰蛋白酶消化之後，在pH值為4左右的情況下 (10-7M)，可以經由Fe-Au探針抓取到許多其胜肽片段，並結合網路上蛋白質資料庫進行比對，可以順利的比對到目標蛋白質。

因為此Fe-Au探針表面具有負電性，在論文中亦嘗試利用Fe-Au探針來改善MALDI-MS中常見分析物訊號抑制的問題：例如在含有干擾物如陰離子型界面活性劑或非離子型界面活性劑的樣品溶液，使用Fe-Au探針在含有干擾物的樣品溶液中抓取到帶有淨正電荷的生化分子，排除干擾物在MALDI-MS分析中抑制分析物訊號的影響。

Gold nanoparticles are used widely in various fields because of their extremely small size, high specific surface areas, and ease of chemical modification. Gold nanoparticles prepared from the reduction of tetrachloroaurate by using trisodium citrate as the reducing agent have negatively charged surfaces because of the attachments of anions such as citrate ions onto their AuI shell surfaces. Based on this characteristic, this thesis demonstrates that gold nanoparticle-based probes are capable of trapping net positively charged analytes from sample solutions. In order to simplify the isolation process of gold nanoparticles from the sample solution, the gold nanoparticles were covalently bond to the surface of magnetic particles through S-Au bonding to generate Fe-Au probes. The Fe-Au particles then could be easily isolated from the sample solution by employing a magnetic field. After isolation, the target species attached on the surfaces of the Fe-Au particles were characterized by using MALDI-MS.

The Fe-Au probes have been successfully used to concentrate trace of cationic surfactants, peptides, and proteins from aqueous samples. By manipulating the pH values of sample solutions, the specific proteins or peptides can be selectively trapped by the Fe-Au probes. The concentrations of protein enzymatic digest products are generally lower than that can be directly detected by using MALDI-MS. The Fe-Au probes can be employed to concentrate the positively charged peptide residues from sample solutions by simply adjusting the pH value of sample solutions to 4, which leads most of the peptide residues carrying net positive-charges. The Fe-Au probes have been successfully used to concentrate a low concentration of cytochrome C (10-7 M) tryptic digest product from a sample solution at pH 4.0. The trapping peptides residues were characterized via the combination of MALDI-MS analysis results and protein database searches.

Additionally, the Fe-Au probes were also applied to solve the suppression effects arising in MALDI-MS analysis. It has been demonstrated that the Fe-Au probes can selectively trap the positively-charged species from the sample solution containing high concentrations of anionic or nonionic surfactants. Thus, the suppression effects caused by the presence of high concentrations of surfactants in MALDI-MS analysis can be reduced.

中文摘要 …………………………………………………………… Ⅰ
英文摘要 …………………………………………………………… Ⅱ
謝誌 …………………………………………………………… Ⅲ
目錄 …………………………………………………………… Ⅳ
圖表目錄 …………………………………………………………… Ⅷ

　　　壹、緒論 …………………………………………………………… 1
　一、 前言……………………………………………………… 1
　二、 基質輔助雷射脫附游離飛行時間質譜法……………… 3
2-1 MALDI的發展……………………………………… 3
2-2 基質的功能與特性………………………………… 4
2-3 樣品脫附游離的機制……………………………… 7
2-4 飛行時間質量分析儀(TOF)………………………… 9
2-4-1 直線式TOF質量分析儀………………………… 9
2-4-2 反射式TOF質量分析儀………………………… 10
2-5 MALDI-MS應用的範圍…………………………… 12
三、
影響MALDI-MS分析中分析物訊號強弱的因素與改善方法………………………………………………………
14
3-1影響分析物訊號的因素……………………………… 14
3-2改善方法…………………………………………… 15
四、 金奈米粒子的特性與應用……………………………… 19
4-1製備方法……………………………………………… 19
4-2基本特性……………………………………………… 20
4-3金奈米粒子表面的修飾方法………………………… 22
　 4-4金奈米粒子的應用…………………………………… 22
五、 磁性粒子………………………………………………… 24
5-1磁性粒子的合成……………………………………… 24
5-2磁性粒子的應用……………………………………… 25
六、 論文目標………………………………………………… 26

　　　貳、實驗 …………………………………………………………… 27
　一、 實驗藥品及材料………………………………………… 27
　二、 實驗儀器………………………………………………… 29
　三、 實驗步驟與流程………………………………………… 30
3-1以金奈米粒子為探針之實驗………………………… 30
3-1-1金奈米粒子的合成方法………………………… 30
3-1-2以表面修飾有金奈米粒子的光纖為探針進行陽
離子型界面活性劑的萃取………………………
30
3-1-3以金奈米粒子為探針濃縮陽離子型界面活性劑 31
3-1-4以金奈米粒子為探針選擇濃縮特定的蛋白質樣
品…………………………………………………
31
3-2表面上修飾金奈米粒子的磁性粒子 (Fe-Au粒子)
為選擇濃縮樣品之探針之實驗………………………
32
3-2-1磁性粒子的合成………………………………… 32
3-2-2 Fe-Au粒子的製備方法………………………… 32
(1) TEOS溶膠凝膠的合成…………………………… 32
(2)在磁性粒子表面修飾溶膠凝膠矽氧化合物……… 32
(3) 在磁性粒子表面修飾含硫醇基的化合物………… 33
(4) 金奈米粒子修飾磁性粒子表面…………………… 33
3-2-3 Fe-Au粒子為探針之實驗……………………… 33
(1) 濃縮陽離子型界面活性劑……………………… 33
(2) 選擇濃縮生化樣品……………………………… 35
(3) 選擇濃縮cytochrome c經胰蛋白酶 (Trypsin) 消
化產物………………………………………………
35
(4) 溶液環境對選擇濃縮特定分析物之影響………… 36
(5) MALDI樣品的製備………………………………… 36
3-3表面修飾四級銨鹽的磁性粒子於吸附負電荷樣品
之實驗…………………………………………………
37
3-3-1選擇特定靜電荷的蛋白質……………………… 37

參、結果 與討論…………………………………………………… 40
一、 以金奈米粒子做為選擇濃縮樣品的探針……………… 40
1-1以表面修飾有金奈米粒子的光纖進行陽離子型界
面活性劑的萃取……………………………………
40
1-2直接以金奈米粒子為探針對陽離子型界面活性劑
進行濃縮之探討……………………………………
46
　　　　　
1-3金奈米粒子應用於生化分子的選擇性濃縮……… 50
二、 以Fe-Au粒子為探針…………………………………… 53
2-1 以Fe-Au粒子為探針進行陽離子型界面活性劑CTAB的濃縮………………………………………
55
2-2 以Fe-Au粒子為探針進行水中微量陽離子型界面活性劑的濃縮………………………………………
60
2-3 以Fe-Au粒子為探針進行胜肽及蛋白質樣品的濃
縮……………………………………………………
64
2-4 Fe-Au粒子應用於蛋白質消化產物的濃縮……… 75
2-5溶液環境對濃縮分析物之影響…………………… 82
三、
表面修飾四級銨鹽的磁性粒子於選擇濃縮特定淨電荷蛋白質的探討……………………………………………
93

　　　肆、結論 …………………………………………………………… 97

參考文獻 …………………………………………………………… 99

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