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研究生:張晏祥
研究生(外文):Yen Hsiang Chang
論文名稱:氧化鐵奈米粒子萃取substanceP應用於基質輔助雷射脫附游離飛行時間質譜法的研究
論文名稱(外文):Extraction of Substance P by Iron Oxide Nanoparticlefor Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
指導教授:張玉珍張玉珍引用關係
指導教授(外文):Yu Chen Chang
學位類別:碩士
校院名稱:東海大學
系所名稱:化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:88
中文關鍵詞:基質輔助雷射脫附游離飛行時間質譜法
外文關鍵詞:Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
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摘要
Substance P係屬神經胜肽(neuropeptide)其中一種,主要分布於中樞神經,許多生理上的反應都與其分泌含量有關。故監控substance P對於生理上的影響相形重要。過去在於分析substance P方法上常利用ELISA Kit搭配酵素免疫分析儀 (ELISA reader)或Radio-immunoassay (RIA)方法,須經過繁複的處理及耗時,同時所需樣品量大。
本研究將經修飾後表面帶負電荷氧化鐵奈米粒子(iron oxide nanoparticle ,IONP@HS-C2-COOH),藉著在特定pH值環境下經由靜電作用力篩選濃縮具正電性的substance P,接著將這些奈米粒子收集並且經過簡單的清洗後混以α-CHCA,直接以基質輔助雷射脫附飛行時間質譜儀(Matrix-assisted Laser Desorption Ionization / Time of Flight Mass Spectrometer, MALDI-TOF MS)進行偵測。此分析方法成功的應用於老鼠脊髓液(spinal cord fluid)。在標準溶液中線性範圍5.0×10-8 ~5.0×10-10 M,偵測極限為9.8x10-11M,老鼠脊髓液樣品經氧化鐵奈米粒子濃縮後,其線性範圍1.0×10-7 ~1.0×10-8 M,偵測極限為3.1x10-9M,回收率可達89%以上。
Extraction of Substance P by Iron Oxide Nanoparticle
for Matrix Assisted Laser Desorption/Ionization Time-of-Flight
Mass Spectrometry
目 錄
中文摘要 I
謝誌 II
目錄 III
圖目錄 VI
表目錄 VIII
壹、緒論 1
ㄧ、前言 1
二、基質輔助雷射脫附游離飛行時間質譜法 2
2-1 MALDI的發展 2
2-2基質的特性 3
2-3 MALDI離子形成的機制 6
2-4飛行時間質量分析儀的原理 8
2-5MALDI-TOF MS的優點 12
2-6影響MALDI-TOF MS的因素 13
2-7奈米粒子應用於MALDI的方法 15
三、Substance P(物質P) 16
3-1 Substance P簡介 16
3-2過去的分析方法 18
四、研究動機 20
貳、實驗 21
ㄧ、藥品 21
二、儀器設備 23
三、實驗方法 23
3-1藥品配製 23
3-2氧化鐵磁性奈米粒子的製備與表面修飾 24
3-3 MALDI樣品的配製 25
3-4 ZipTip® Pipette Tips去陽離子處理 26
3-5真實樣品界面活性劑緩衝溶液配製 27
3-6樣品盤(target)前處理 27
3-7質譜儀器操作條件 28
參、結果與討論 30
ㄧ、氧化鐵奈米粒子(IONP)鑑定與特性分析 30
二、Substance P最佳吸附條件之研究 32
2-1疏水性碳鏈粒子比較 32
2-2 pH值探討 34
2-3 IONP@HS-C2-COOH添加量之研究 37
2-4 IONP@HS-C2-COOH萃取後清洗 39
三、以IONP@HS-C2-COOH為濃縮探針之探討 41
四、生物臨床檢體中干擾物的研究 45
五、Substance P檢量線製作 52
六、脊髓液樣品的分析 56
七、老鼠脊髓液樣品中substance P檢量線的製作 56
八、回收率的探討 60
九、老鼠脊髓液樣品定量 61
肆、結論與未來展望 63
參考文獻 64







圖目錄
圖(一)直線型及反射型飛行時間質譜儀 11
圖(二) Substance P結構與pKa 17
圖(三) 實驗流程圖 26
圖(四) TEM粒徑分佈圖 31
圖(五) 氧化鐵奈米粒子(IONP)表面修飾不同碳鏈數粒子與上清液質
譜圖訊號強度比較 33
圖(六) Substance P在不同pH值經ION@HS-C2-COOH吸附後粒
子與上清液之質譜圖訊號強度比較 35
圖(七) ION@HS-C2-COOH添加量對substance P訊號影響的比較
圖 38
圖(八) Substance P與Angiotensin I 配製於磷酸緩衝溶液中比較經
ION@HS-C2-COOH萃取水洗次數之質譜圖 40
圖(九) Substance P配製於磷酸緩衝溶液中以ION@HS-C2-COOH萃
取之質譜圖 42
圖(十) Substance P配製於磷酸緩衝溶液中以不同處理方法之質譜圖 44
圖(十一) Substance P配製於界面活性劑緩衝溶液中以IONP@
HS-C2-COOH萃取未抑制1300Da以下質量偵測訊號之質譜
圖 46
圖(十二) Triton® X-100以DHB基質之質譜圖 47
圖(十三) Substance P配製於界面活性劑緩衝溶液ION@HS-C2-COOH
萃取抑制1300Da以下質量偵測訊號之質譜圖 50
圖(十四) Substance P配製於界面活性劑緩衝溶液以ION@ HS-C2
-COOH萃取抑制1300Da以下質量偵測訊號之質譜圖 51
圖(十五) 脊髓液中substance P之質譜圖 59
圖(十六) 老鼠脊髓液樣品質譜圖 62





















表目錄
表(一) MALDI基質及適用的雷射波長和分析物 4
表(二) Substance P在不同組織或細胞中的作用 18
表(三)MALDI-TOF MS操作參數設定 29
表(四) ION@HS-C2-COOH與ZipTip® SCX未萃取、萃取訊號抑制後
訊號/雜訊比(S/N) 49
表(五) IONP@HS-C2-COOH萃取後MALDI-TOF再現性 53
表(六) 標準水溶液與界面活性劑緩衝溶液檢量線 55
表(七) Substance P在脊髓液樣品中的分析檢量線與偵測極限 58
表(八) 老鼠脊髓液中substance P之回收率 60
表(九) 老鼠脊髓液樣品定量結果 61
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