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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張煥宗(Huan-Tsung Chang) | |
| dc.contributor.author | Yu-Yen Kao | en |
| dc.contributor.author | 高毓言 | zh_TW |
| dc.date.accessioned | 2021-06-15T01:29:07Z | - |
| dc.date.available | 2010-07-24 | |
| dc.date.copyright | 2009-07-24 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-07-21 | |
| dc.identifier.citation | 第一章參考文獻
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42926 | - |
| dc.description.abstract | 本論文之主要目的為發展微胞電動力毛細管層析(micellar electrokinetic chromatography, MEKC)結合發光二極體誘導螢光偵測法(light-emitting diode-induced fluorescence detection)分析經naphthalene-2,3-dicarboxaldehyde (NDA)衍生化的胺基酸(amino acids)以及生物胺(biogenic amines)。藉由探討緩衝液的pH值及所含之十二烷基硫酸鈉(sodium dodecyl sulfate, SDS)、聚氧化乙烯(poly(ethylene oxide), PEO)和Tris-borate (TB)濃度對分離效率與解析度之影響，發現最佳化的分離條件為：用含40 mM SDS的0.5 M TB (pH 10.2)緩衝溶液來填充毛細管，及使用含35 mM SDS之100 mM TB (pH 9.0)緩衝溶液來配製0.1% PEO。在此條件下，我們可於16分鐘內成功分離14種胺類小分子，其偵測極限(S/N = 3)從2.1 nM甘胺酸(glycine, Gly)到19.2 nM血清素(serotonin, 5-HT)。分析物遷移時間的相對標準偏差(relative standard deviation, RSD)皆小於0.5% (n = 5)。應用此分離技術，分別對乳癌細胞(MCF-7)與正常乳房表皮細胞(H184B5F5/M10) (2-3 × 106 cells/mL)進行分析(三次測量)，並定量細胞中9種(MCF-7)以及10種(H184B5F5/M10)胺基酸，包括絲胺酸(serine, Ser)、麩醯胺酸(glutamine, Gln)、組胺酸(histidine, His)、Gly、丙胺酸(alanine, Ala)、牛磺酸(taurine, Tau)、酪胺酸(tyrosine, Tyr)、麩胺酸(glutamate, Glu)、纈胺酸(valine, Val)、天冬胺酸(aspartate, Asp)。發現兩種細胞在部分胺基酸含量上確實有差異，推測部分原因與癌細胞需要大量分化生殖，消耗能量有關。我們也於細胞繼代後不同時間，收集細胞去做胺基酸分析，並計算單一細胞胺基酸的含量，癌細胞由於生長速率較快，經過48及72小時後，觀察到部份胺基酸是明顯減少的，反觀正常細胞，則因為生長速度緩慢，胺基酸消耗的速度較不明顯。
為了提高偵測極限，我們利用相同的緩衝溶液條件與毛細管處理方式，進行線上濃縮與分離胺基酸與生物胺。在電壓驅動下，經NDA衍生化的分析物進入到含有SDS的PEO溶液當中，會因樣品區帶和PEO溶液之間電導度與黏度的差異，進行線上濃縮與分離。藉由探討樣品區帶中TB的濃度和添加氯化鈉(NaCl)的量對濃縮效果的影響，發現樣品濃縮倍率可由4.4倍(His)至38.7倍(Glu)，其偵測極限(S/N = 3)從0.10 nM (Glu)到3.63 nM (5-HT)。利用此線上濃縮與分離技術，分析0.284-μL 之乳癌細胞(MCF-7) (2 × 105 cells/mL)，並定量細胞中9 種胺基酸，得到的結果與前一部份使用單純分離的方法是相近的。 | zh_TW |
| dc.description.abstract | We have demonstrated separation of the derivatives of amino acids and biogenic amines with naphthalene-2,3-dicarboxaldehyde (NDA) by micellar electrokinetic chromatography (MEKC) in conjunction with a purple light-emitting diode-induced fluorescence detection using poly(ethylene oxide) (PEO). The roles that the concentrations of PEO, sodium dodecyl sulfate (SDS), and Tris-borate (TB) at various pH values played in resolution and separation efficiency of the analytes have been investigated. After injecting the sample to a capillary filled with 0.5 M TB buffer (pH 10.2) containing 40 mM SDS, 0.1% PEO that has been prepared in 100 mM TB (pH 9.0) containing 35 mM SDS enters the capillary. This MEKC condition allows separation of 14 analytes within 16 minutes, with limits of detection (LODs) at signal-to-noise ratio (S/N) of 3 ranging from 2.06 nM (glycine, Gly) to 19.2 nM (serotonin, 5-HT). RSD values (n = 5) of the migration times for the analytes are all less than 0.5%. This approach has been validated for the analysis of human breast cancer cells (MCF-7) and normal epithelial cell line (H184B5F5/M10) (2-3 × 106 cells /mL). The amounts of 9 (MCF-7) or 10 (H184B5F5/M10) amino acids, including serine (Ser), glutamine (Gln), histidine (His), Gly, alanine (Ala), taurine (Tau), tyrosine (Tyr), glutamate (Glu), valine (Val), and aspartate (Asp) in one cell could be calculated, respectively. The amounts of some amino acids in MCF-7 and H184B5F5/M10 are significantly different, maybe because of proliferation of cancer cells which consume much energy. Besides, we calculated the amounts of amino acids in cells incubated at different time interval, and found that the consumption of some amino acids in cancer cells rather than normal cells was obvious.
In order to further improve the sensitivity of the amino acids and biogenic amines, we developed an on-line concentration technique using similar MEKC conditions. Once high voltage is applied, the NDA derivatives enter PEO solution containing SDS and then become stacked due to decreases in electric field and increases in viscosity. We have also found that TB and sodium chloride in the sample zone play some roles in determining the stacking efficiency. This on-line concentration MEKC technique allows improvement in the detection of the analytes by factors ranging from 4.4 (His) to 38.7 (Glu), leading to their LODs ranging from 0.10 nM (Glu) to 3.63 nM (5-HT). This approach has been validated by the analysis of 0.284-μL MCF-7 cells (2 × 105 cells/mL), and the results were consistent with those described in the first part. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T01:29:07Z (GMT). No. of bitstreams: 1 ntu-98-R96223206-1.pdf: 1136642 bytes, checksum: ee98aa6f8ca0844b60727736d3cfe4da (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 目 錄
中文摘要………………………………………………………………………………I 英文摘要……………………………………………………………………………..III 目錄…………………………………………………………………………………...V 表目錄……………………………………………………………………………...VIII 圖目錄……………………………………………………………………………......X 第一章 毛細管電泳之概述…………………………………………………………1 1.1 毛細管電泳發展歷史………………………………………………………….1 1.2 毛細管電泳原理……………………………………………………………….2 1.2.1 電淌度……………………………………………………………………..2 1.2.2 電滲流與zeta電位………………………………………………………..3 1.2.3 電泳層析圖………………………………………………………………..5 1.2.4 分離解析度………………………………………………………………..7 1.2.5 分離效率…………………………………………………………………..7 1.2.6 影響分離效率的因素……………………………………………………..8 1.2.6.1 焦耳熱……………………………………………………………...…8 1.2.6.2 樣品吸附………………………………………………………….......9 1.2.6.3 進樣長度………………………………………………………….......9 1.3 毛細管電泳分離機制…………………………………………………….......10 1.3.1 微胞電動力毛細管層析法……………………………………………....10 1.4 偵測極限與線上濃縮……………………………………...…………………12 1.4.1 場放大堆積………………………………………………………………12 1.4.2 流動式聚合物濃縮與分離生物分子……………………………………14 1.4.3 掃掠樣品堆積法…………………………………………………………15 1.5 毛細管電泳之偵測系統…………………………………………………...…17 1.5.1雷射激發誘導螢光……………………………………………………….17 1.6 研究動機……………………………………………………………………...18 1.7 參考文獻……………………………………………………………………...20 第二章 毛細管電泳之胺類小分子分離及細胞分析……………………………..23 2.1 前言…………………………………………………………………………...23 2.2 實驗…………………………………………………………………………...29 2.2.1 實驗藥品………………………………………………………………....29 2.2.2 標準品、緩衝溶液與細胞培養液的製備……………………………....29 2.2.3 細胞培養方法…………………………………………………………....30 2.2.4 細胞計數與細胞均質液的製備………………………………………....30 2.2.5 胺基酸衍生化…………………………………………………………....30 2.2.6 毛細管電泳系統………………………………………...…………….....31 2.2.7 發光二極體誘導螢光偵測系統…………………………………………31 2.2.8 毛細管電泳分析………………………………………...…………….....32 2.3 結果與討論…………………………………………………………………...32 2.3.1 SDS與PEO的濃度對分析物遷移時間的影響………………………...32 2.3.2 充填毛細管的緩衝溶液中TB與SDS對分離的影響…………...…....39 2.3.3 分離緩衝溶液中SDS, PEO, TB濃度和pH對於分離的影響……………………………………………………………..…….......44 2.3.4 分離14種NDA衍生化的胺類小分子…………………………...…...52 2.3.5 細胞之胺基酸與生物胺分析………………...…………………...…….57 2.3.6 細胞於不同生長時期胺基酸含量的變化…...……………………..…..61 2.4 結論………………………………………………………………..................63 2.5 參考文獻…………………………………………………………..................65 第三章 非連續態電泳系統線上濃縮胺類小分子與乳癌細胞分析….................69 3.1 前言…………………………………………………………………...……....69 3.2 實驗…………………………………………………………………………...71 3.2.1 實驗藥品………………………………………………………………....71 3.2.2 標準品、緩衝溶液與細胞培養液的製備……………………………....71 3.2.3 細胞培養方法…………………………………………………………....71 3.2.4 細胞計數與細胞均質液的製備………………………………………....71 3.2.5 胺基酸衍生化…………………………………………………………....71 3.2.6 毛細管電泳系統………………………………………...…………….....71 3.2.7 發光二極體誘導螢光偵測系統…………………………………………71 3.2.8 線上濃縮方法…………………………………………………………....71 3.3 結果與討論…………………………………………………………………...72 3.3.1 樣品區帶之TB濃度對五種分析物線上濃縮效果的影響………..…...72 3.3.2 十種分析物之線上濃縮以及樣品區帶基質因素探討………………....78 3.3.2.1 樣品區帶TB濃度對十種分析物線上濃縮效果的影響…………....80 3.3.2.2 樣品區帶NaCl濃度對十種分析物線上濃縮效果的影響……….....80 3.3.3 線上濃縮胺類小分子………………………………………………........90 3.3.4 利用線上濃縮系統進行乳癌細胞之胺基酸與生物胺分析………........90 3.4 結論………………………………………………………………...................93 3.5 參考文獻…………………………………………………………...................94 表目錄 Table 1.1 Physical properties of surfactants…………………………………………………11 Table 2.1 Physical and chemical properties of 14 analytes………………………………………………………28 Table 2.2 PEO dependence of the mobility of Tyr and 5-HT in the presence of SDS in separation buffer.…………………………………….38 Table 2.3 Effect of the concentration of SDS in buffer on resolution………………………………………………….39 Table 2.4 Effect of the concentration of SDS in buffer used to fill the capillary on peak height, theoretical plates (N), and resolution………..……………………43 Table 2.5 Effect of the concentration of SDS in separation buffer on peak height, theoretical plates (N), and resolution..……………47 Table 2.6 Effect of the concentration of TB in separation buffer on mobility, theoretical plates (N), and resolution……………………………………………….….51 Table 2.7 Effect of concentration of SDS in separation buffer on peak height and theoretical plates (N) when using 65 cm-capillary to separate 10 analytes……………54 Table 2.8 Effect of concentration of SDS in separation buffer on resolution when using 65 cm-capillary to separate 10 analytes.…………………………………………………54 Table 2.9 Linearities and detection limits in the determination of 14 analytes.………………………………56 Table 2.10 Analytical results of lysate for MCF-7 cells………………………………………………………………61 Table 2.11 The amounts of amino acids in MCF-7 cells under different conditions (fmol per cell)…………………….63 Table 2.12 The amounts of amino acids in H184B5F5/M10 cells under different conditions (fmol per cell) ……63 Table 3.1 Effect of concentration of TB (pH 9.0) in sample zone on peak height, theoretical plates (N), and resolution when using 40 cm-capillary.……….…………76 Table 3.2 Effect of concentration of TB (pH 8.3-8.5) in sample zone on peak height, theoretical plates (N), and resolution when using 40 cm-capillary….…………………77 Table 3.3 Effect of concentration of TB (pH 9.0) in sample zone on peak height, theoretical plates (N) when using 65-cm capillary..……………………….………….….83 Table 3.4 Effect of concentration of TB (pH 9.0) in sample zone on resolution when using 65-cm capillary………………………………………………..…………83 Table 3.5 Effect of concentration of NaCl in sample zone on peak height, theoretical plates (N)………… ………..85 Table 3.6 Effect of concentration of NaCl in sample zone on resolution……….…………………………………………….85 Table 3.7 Effect of NaCl in sample zone containing 20 mM TB on Peak height, theoretical plates (N)………..………87 Table 3.8 Effect of NaCl in sample zone containing 20 mM TB on resolution……………………………………………………87 Table 3.9 Effect of TB in sample zone containing 10 mM NaCl on peak height, theoretical plates (N)………………………………..……………………………………...89 Table 3.10 Effect of TB in sample zone containing 10 mM NaCl on resolution……………………………………………………89 Table 3.11 LOD values for the analytes under different conditions…..……………………………………………………….91 Table 3.12 Calculated recoveries and amounts of amino acids in MCF-7 cells (n = 3) ………………………………….93 圖目錄 Figure 1.1 (A) Schematic representation of proton dissociation on the silica wall; (B) Illustration of double layer and zeta potential………….……………….….………..5 Figure 1.2 Flow profiles of CE (A) and HPLC (B)………………………………………………………………………….6 Figure 1.3 Schematic representation of field amplified sample stacking……….………………………………………………13 Figure 1.4 Schematic representation of sample stacking and separation using polymer solution………………….………15 Figure 1.5 Evolution of micelles and neutral analyte molecules during sweeping in the presence of high electroosmotic flow..………………………………………………16 Figure 2.1 Derivatization process of primary amino acid with NDA in the presence of cyanide.……..…….……………26 Figure 2.2 The structures of 14 analytes.….……………27 Figure 2.3 Instrumental setup of capillary electrophoresis coupled with light-emitting diode-induced fluorescence..………….……………………..……………………32 Figure 2.4 Effect of SDS concentration in TB solution (pH 10.2) that was filled in the capillary on separation of five analytes……...…….…….….………………………………35 Figure 2.5 Effect of SDS concentration in TB solution (pH 10.2) that was filled in the capillary on separation of five analytes when 10 mM SDS in separation buffer.……………………………………….…………………………………36 Figure 2.6 Effect of SDS concentration in TB solution (pH 10.2) that was filled in the capillary on separation of five analytes when 20 mM SDS in separation buffer………………………………………………………………..37 Figure 2.7 Reduced viscosity of PEO as a function of SDS concentration………………………………………………………39 Figure 2.8 Effect of the concentration of TB (pH 10.2) buffer containing 30 mM SDS that was filled in the capillary on the separation of five analytes………………41 Figure 2.9 Effect of SDS concentration in 0.5 M TB (pH 10.2) that was filled in the capillary on the separation of five analytes………………………………………………………42 Figure 2.10 Effect of SDS concentration in 0.1% PEO solution on the separation of five analytes……….…….46 Figure 2.11 Effect of concentration of PEO in 100 mM TB (pH 9.0) containing 30 mM SDS on the separation of five analytes………………………………………………………………48 Figure 2.12 Effect of pH on the separation of analytes………………………………………………………………49 Figure 2.13 Effect of the concentration of TB in 0.1% PEO solution containing 30 mM SDS on the separation of analytes…………….….……………………………………………50 Figure 2.14 Analysis of a mixture containing 10 standard analytes by CE-LEDIF using a 40-cm capillary………………53 Figure 2.15 Analysis of a mixture containing 10 standard analytes by CE-LEDIF using a 65-cm capillary………………53 Figure 2.16 Analysis of a mixture containing 14 standard analytes by CE-LEDIF using a 65-cm capillary………………55 Figure 2.17 Electropherogram of NDA derivatization result of (A) 4th; (B) 5th; (C) 6th PBS used to wash MCF-7 cells…………………………………………………………………59 Figure 2.18 Electropherograms of the lysate of MCF-7 cells with NDA derivatization.…….…………………..………….60 Figure 2.19 Electropherograms of the lysate of H184B5F5/M10 cells with NDA derivatization.…………….60 Figure 3.1 Evolution of stacking and separation of NDA-amino acid derivatives by CE-LEDIF in the presence of EOF and PEO solutions………………………………………………70 Figure 3.2 Effect of the concentration of TB (pH 9.0) in sample zone on stacking of NDA amino acids and amine derivatives by CE-LEDIF.………………………………………74 Figure 3.3 Effect of the concentration of TB (pH 8.3) in sample zone on stacking of NDA amino acids and amine derivatives by CE-LEDIF…………………………………………75 Figure 3.4 On-line concentration of 10 analytes under two conditions……………………………………………………………79 Figure 3.5 Effect of the concentration of TB (pH 9.0) in sample zone on stacking of analytes…………………………82 Figure 3.6 Effect of NaCl in sample zone on stacking of analytes….…………………………………………………………84 Figure 3.7 Effect of NaCl in sample zone containing 20 mM TB (pH 9.0) on stacking of analytes…………………………86 Figure 3.8 Effect of concentration of TB in sample zone containing 10 mM NaCl on stacking of analytes..…………88 Figure 3.9 Analysis of the lysate of MCF-7 cells with on-line concentration…………………………………………………92 | |
| dc.language.iso | zh-TW | |
| dc.title | 乳癌細胞中胺基酸與生物胺之微胞電動力毛細管層析分析 | zh_TW |
| dc.title | Analysis of Amino Acids and Biogenic Amines in Breast Cancer Cells by Micellar Electrokinetic Chromatography | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 劉春櫻(Chuen-Ying Liu),吳秀梅(Shou-Mei Wu),邱泰嘉(Tai-Chia Chiu) | |
| dc.subject.keyword | 微胞電動力毛細管層析,發光二極體誘導螢光偵測法,胺基酸,生物胺,十二烷基硫酸鈉,聚氧化乙烯,乳癌細胞, | zh_TW |
| dc.subject.keyword | Micellar electrokinetic chromatography,Light-emitting diode induced fluorescence detection,Amino acids,Biogenic amines,Sodium dodecyl sulfate,Poly(ethylene oxide), | en |
| dc.relation.page | 94 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-07-22 | |
| dc.contributor.author-college | 理學院 | zh_TW |
| dc.contributor.author-dept | 化學研究所 | zh_TW |
| 顯示於系所單位： | 化學系 | |
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