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研究生: 林鐘偉
Chung-wei Lin
論文名稱: 結合一奈米結構(nanofilament silicon , nSi)矽基材與表面化學改質之質譜晶片用以提升質譜效率之研究
The study of sensitivity improvement of mass spectrometry by nanofilament silicon substrate (nSi) and surface chemical modification
指導教授: 陳文逸
Wen-yih Chen
口試委員:
學位類別: 碩士
Master
系所名稱: 工學院 - 化學工程與材料工程學系
Department of Chemical & Materials Engineering
畢業學年度: 98
語文別: 中文
論文頁數: 107
中文關鍵詞: 質譜晶片表面化學改質奈米結構矽基材
外文關鍵詞: mass spectrometry, surface chemical modification, nanofilament silicon substrate
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  • 摘要
    傳統在複雜蛋白質的分離以及定序的分析技術中有著儀器設備昂貴,分析時間過長,分離條件限制較多,分離產物不純等等問題,而近年來質譜技術的進步,可改善這個問題。
    質譜儀是偵測質荷比(mass to charge ration, m/z)來決定分析物分子量的技術,相較於其他的質譜分析有著基質(matrix)干擾與分析物帶有多電荷等問題,本實驗發展新一代高靈敏奈米層狀矽基材 (nanofilament Silicon , nSi)質譜分析晶片來改善這些問題。進一步本研究利用表面改質之方式將nSi質譜分析晶片分別處理成疏水性(hydrophobic)、親水性(hydrophilic)、正電性(cation exchange)、負電性(anion exchange)、固定化金屬親和性 (IMAC) 等不同表面特性的質譜分析晶片。如此 nSi 質譜分析晶片表面能對分析物做純化與分離,進而增加質譜分析之選擇性 (selectivity) 以及靈敏度 (sensitivity)。
    從實驗中,質譜的分析數據以及表面的化學鑑定來看,本研究所建構之nSi質譜分析晶片:
    1. 隨著蝕刻秒數之增加,奈米結構的表面變較粗糙,使得質譜訊號隨之提升,但卻有一定之極限。
    2. 不同表面特性之晶片在經過化學分析電子儀(ESCA)分析後,得到其相對應之元素訊號(如:在疏水性晶片上所得到之碳訊號)比之未經過化學改質之晶片所得到之訊號都有增加。
    3. 不同特性之胜肽在其相對應的表面所得之質譜測試訊號,比之未經過化學改質之晶片都有明顯之提升。


    Abstract
    Some problems exist in the conventional separation of complex protein mixtures and the sequence analysis technique. These problems include expensive equipment, extremely long analysis time, restrictive separation conditions, and impure separation results. Employing advanced mass spectrometry techniques improves these issues.
    Mass spectrometry is a technology that determines the molecular weight of the analyte by detecting its mass-to-charge ratio. Compared to other mass spectrometry methods that has matrix interference and analyte with multiple charges. To develop the new nSi mass-analytical chip will improve these problems. This study utilizes surface modification to process the chip surface and forms different nSi mass-analytical chip, such as hydrophobic、hydrophilic、cation exchange、anion exchange、and metal affinity (IMAC). This approach enables analyte purification and separation, increasing the selectivity and sensitivity of mass spectrometric analysis.
    According to mass spectrometry (MS) analytic data and chemical surface identification in the experiment, the nSi mass-analytical chip established in this study was characterized as the following:
    1. As the etching time increased, the nano-structured surface of the mass spectrometric analysis chips became coarser. MS signal intensity subsequently increased, but remained under a specific level.
    2. After the analysis of ESCA (electron spectroscopy for chemical analysis), chips with different surface properties obtained more corresponding element signals (e.g., a carbon signal received on a hydrophobic chip) than chips without chemical modification.
    3. Chips after chemical modification with different properties peptides could get higher signal intensity than chips without chemical modification.

    目錄 摘要 i Abstract iii 誌謝 v 目錄 vi 圖目錄 ix 表目錄 xiii 一、 緒論 1 二、文獻回顧 3 2.1質譜儀的發展起源 3 2.2質譜離子源介紹 5 2.2.1 電噴灑離子化法 6 2.2.1.1電噴灑離子源構造 6 2.2.1.2 電噴灑如何形成氣態離子 7 2.2.1.3電噴灑離子化法的優缺點 11 2.2.2 基質輔助雷射脫附離子化法 13 2.2.2.1基質的特性及作用 15 2.2.2.2 基質輔助雷射脫附離子化法的離子化機制 17 2.2.2.3 基質輔助雷射脫附離子化法的優缺點 18 2.2.3 表面輔助雷射脫附離子化 20 2.2.3.1 碳粉 21 2.2.3.2 奈米碳管 21 2.2.3.3 金奈米粒子 22 2.2.3.4 氧化鐵微米粒子 22 2.2.3.5 二氧化鈦奈米粒子 23 2.2.3.6 Desorption/ionization mass spectrometry on porous silicon 24 2.2.3.7 矽材及其衍生物 26 2.3 表面增強雷射脫附離子化 27 三、實驗材料與方法 31 3.1實驗設備 31 3.2 實驗藥品 32 3.3 實驗方法 34 3.3.1 矽晶片表面蝕刻 34 3.3.2 表面化學改質 35 3.3.3 質譜儀測試 41 3.3.4 螢光驗證 43 四、結果與討論 44 4.1 矽晶片表面結構最佳化之建構 44 4.2 矽晶片不同表面之改質與鑑定 51 4.2.1 不同化學表面之鑑定 52 4.2.2 固定化金屬離子親和表面之鑑定 56 4.2.3 矽晶片表面化學改質之質譜分析 57 4.2.3.1親疏水性表面質譜結果 59 4.2.3.2正負電性表面質譜結果 65 4.2.3.3固定化金屬離子親和表面質譜結果 68 五、結論 77 參考文獻 79 Acknowledgement 90 圖目錄 圖2. 1 質譜儀結構圖 4 圖2. 2 電噴灑離子化構造與質譜儀銜接介面 7 圖2. 3 (A)溶液中解離的正離子受電場牽引,推擠管道出口端液面成為圓錐形(B)正離子受電場牽引之力大於液面的表面張力,形成可穩定產生電噴灑的泰勒錐 8 圖2. 4 電噴灑所產生的帶電荷液滴如何形成氣相離子的過程示意圖 9 圖2. 5 基質輔助雷射脫附離子化法示意圖 14 圖2. 6 不同化學性質或固定不同生物分子之晶片表面 30 圖3. 1 矽晶片表面蝕刻流程圖 34 圖3. 2 疏水性表面官能基改質法 35 圖3. 3 親水性官能基表面改質法 37 圖3. 4 正電性官能基表面改質法 38 圖3. 5 負電性官能基表面改質法 38 圖3. 6 帶有金屬離子的IMAC表面改質法 40 圖3. 7 質譜分析流程圖 42 圖4. 1 沉積金薄膜厚度3 nm矽基材表面SEM圖 45 圖4. 2 輔助蝕刻金屬為金、沉積厚度3 nm、蝕刻溶液為HF/ Ethanol/ H2O2(1:1:1 V/V/V)所做的不同蝕刻時間作質譜效率測試,樣本為Des-Arg9- Bradykinin、濃度10-7 M 46 圖4. 3 輔助蝕刻金屬為金、沉積厚度3 nm、蝕刻溶液為HF/ Ethanol/ H2O2(1:1:1 V/V/V)所做的不同蝕刻時間作質譜效率測試,樣本為Des-Arg9- Bradykinin、濃度10-8 M 46 圖4. 4 輔助蝕刻金屬為金、沉積厚度3 nm、蝕刻溶液為HF/ Ethanol/ H2O2(1:1:1 V/V/V)所做的不同蝕刻時間作質譜效率測試,樣本為Des-Arg9- Bradykinin、濃度10-9 M 47 圖4. 5 輔助蝕刻金屬為金、沉積厚度3 nm、蝕刻溶液為HF/ Ethanol/ H2O2(1:1:1 V/V/V)樣本為Des-Arg9- Bradykinin之不同蝕刻時間的訊號/雜訊比(S/N ratio) 47 圖4. 6不同蝕刻時間之質譜測試圖(a) 30 s (b) 60 s (c) 180 s (d) 300 s (e) 360 s 49 圖4. 7 輔助蝕刻金屬為金、沉積厚度3 nm、蝕刻溶液為HF/ Ehanol/ H2O2(1:1:1 V/V/V)樣本為Des-Arg9- Bradykinin之不同蝕刻時間對螢光測試的結果,其中(a) 蝕刻時間30秒之矽晶片(b) 蝕刻時間60秒之矽晶片(c) 蝕刻時間180秒之矽晶片(d) 蝕刻時間300秒之矽晶片(e) 蝕刻時間360秒之矽晶片 50 圖4. 8 不同表面性質之矽晶片示意圖。 51 圖4. 9 疏水性表面之化學分析電子儀分析圖 52 圖4. 10 親水性表面之化學分析電子儀分析圖 53 圖4. 11 正電性表面之化學分析電子儀分析圖 54 圖4. 12 負電性表面之化學分析電子儀分析圖 55 圖4. 13 固定化金屬離子親和表面(IMAC)之化學分析電子儀分析圖 56 圖4. 14 (a) 疏水性胜肽及(b)親水性胜肽在不同化學性質表面之質譜測試圖 61 圖4. 15 疏水性胜肽在不同特性表面之質譜圖 (a)純蝕刻之表面 (b) 疏水性表面 (c) 正電性表面 (d) 負電性表面 (e) 銅離子表面 (f) 鎳離子表面 63 圖4. 16 親水性胜肽在不同特性表面之質譜圖 (a) 純蝕刻之表面(b) 疏水性表面 (c) 負電性表面 64 圖4. 17 正電性胜肽在不同化學性質表面之質譜測試圖 66 圖4. 18 正電性胜肽在不同特性表面之質譜圖 (a) 純蝕刻之表面 (b) 疏水性表面 (c) 負電性表面 67 圖4. 19含有連續三個Histidine殘基之胜肽在不同化學性質表面之質譜測試圖 70 圖4. 20 含連續三個Histidine殘基之胜肽在不同特性表面之質譜圖 (a) 純蝕刻之表面 (b) 疏水性表面 (c) 負電性表面 (d) 銅離子表面 (e) 鎳離子表面 72 圖4. 21 含有一個Histidine殘基之胜肽在不同化學性質表面之質譜測試圖 74 圖4. 22 含一個Histidine殘基之胜肽在不同特性表面之質譜圖 (a) 純蝕刻之表面 (b) 疏水性表面 (c) 負電性表面(d) 銅離子表面 (e) 鎳離子表面 76 表目錄 表2. 1 常用的基質(Matrix)及適用的分析物 16 表4. 1 蝕刻時間與表面粗糙度之關係 49 表4. 2 本實驗用各胜肽基本特性 58 表4. 3 疏水性胜肽以及親水性胜肽在不同化學性質表面之質譜訊號值 61 表4. 4 正電性胜肽在不同化學性質表面之質譜訊號值 66 表4. 5 含有連續三個Histidine殘基之胜肽在不同化學性質表面之質譜訊號值 70 表4. 6 含有一個Histidine殘基之胜肽在不同化學性質表面之質譜訊號值 74

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