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研究生:方韻淑
研究生(外文):Fang,Yun-Shu
論文名稱:電泳晶片之表面修飾以抑制擴散及提昇分離效率
論文名稱(外文):Surface Modification to Suppress Dispersion and Enhance Separation for Electrophoresis Microchip
指導教授:陳壽椿
指導教授(外文):Show-Chuen Chen
學位類別:碩士
校院名稱:輔仁大學
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:2010
畢業學年度:98
語文別:中文
論文頁數:123
中文關鍵詞:PEO濃度梯度酸鹼梯度pH梯度表面修飾電滲透流微電泳晶片毛細管電泳
外文關鍵詞:capillary electrophoresisμ-CEelectroosmotic flow (EOF)surface modificationpH gradientPEO concentration gradient
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本文主研究水溶性高分子以及流道表面 pH 值對於微電泳晶片效能的影響。使用此兩種修飾方法對於改變流道表面矽醇基Si-OH解離程度具有方便性易控制性等優點。使用高分子修飾於流道管壁的方式已使用多年,本實驗有別於一般傳統分離DNA的方式─形成凝膠聚合物分離,高於數千個數量級的濃度。然而,現在有一種直接使用的方式在毛細管電泳中使用低於1ppm的濃度。使用500萬分子量的PEO透過氫鍵鍵結在流道內,可長時間的運作。聚環氧乙烷(PEO)使用0.5ppm水溶液在微電泳晶片流道中,可抑制電擴散的效應。並有助於降低前驅物多巴胺的各項實驗數據。
透過表面pH值及稀鹽酸來減少表面表面矽醇基(Si-OH)解離程度,除非表面梯度需要很少使用鹼來增加EOF,因為使用鹼在微電泳晶片中式基本的。
使用 HCl 可用來調整流道表面矽醇基(Si-OH)解離程度,適當添加鹼性溶液於流道表面加速流道的 EOF 仍是必要的。因此以 pH在流道表面建立梯度至至為重要的。
實驗所建立流道梯度經過連續 5 小時的量測,滯留時間的標準偏差 < 1% ,半高寬的變化在2 % 間,可顯示出其穩定性十分良好,而建立檢量線範圍在 20至 800 μM。
然而,在等度的pH或PEO中較晚分離出的樣品的滯留時間、半高寬比較容易產生較大的偏差結果,分離結果的品質不佳。若單只使用一般的層析方式無法解決此問題。此種困擾可以透過在微電泳晶片中建立梯度來解決。透過緩衝溶液或聚環氧乙烷(PEO)於流道表面建立一線性梯度,透過濃度或時間的控制在模擬梯度的視窗中建立最佳化梯度。
理想表面Si-O-覆蓋程度,使用 0.01 M的 HCl 以及 0.5 ppm 的 PEO 沖提於十字流道,用於減少 Si-O-的分布密度,降低電擴散的效應,流道末端沖提 0.1M 的 NaOH 使末端Si-O-分布密度提昇,加速移動較慢的樣品層析峰。並透過高壓時序控制器控制連續實驗步驟以及溶液的更換。
實驗中所建立流道表面Si-O-的梯度,提升最高樣品靈敏度達 30% 以上, R2 可從未修飾的 0.990 提升至 0.995以上。
This study explores how charging traces of hydrophilic polymer, PEO, onto the surface or changing the pH there may improve the capillary electrophoresis’s efficiency. Both efforts enjoy the convenience of tailoring the surface density of silanolate groups, Si-OH, to regulate the migration speed. The presence of trace PEO, an old trick to quiet the turbulence in engineering, works on the stabilizing principle. It is different from the sieving mechanism popular in the DNA analysis--made possible by the formation of a gelled polymeric matrix. The concentration is a few thousand orders of magnitude higher. However, direct transfer of the method can be disastrous, for one, the useful concentration range for CE lies only at or below 1 ppm. The string of H-bonded beads of the macromolecule of the size of 5 million daltons and the sparing solubility provide anchorage for the macro strand to survive continual long hours of service. Coverage of polyethylene oxide, PEO, by driving a 0.5-ppm aqueous solution through the micro CE channel suppresses electrodispersion at the junction. It also helps to slow down the forerunners such as dopamine to gain time to resolve among them.
Trimming the silanolate groups by tuning down the surface pH with dilute HCl solutions serves the same purpose. The effort to alkalify the surface to quicken EOF is seldom necessary unless a gradient pH surface is in need, since it is intrinsically basic when made.
Either adjustment endures a 5-hour service test with deviations below 1% in migration time and about 2% in peak width at half height. The calibration curve is linear over the range between 20 and 80 μM.
However, the late emergent components from an isocratic surface in pH or PEO suffer noticeable deviations in migration time, peak height and peak width. The analytical quality is at stake. Gradient migration can be the answer, but is it workable with micro CE Implementation is difficult by the usual chromatographic means. When a buffer or a PEO solution flows over the silanolate surface it forms a sigmoid front that also inbreeds a linear gradient portion--much as an old solution awaits a new problem. The question is to find the right time window to bracket the linear gradient portion to span the separation channel and to locate the optimal concentration of the feeding solution.
The ideal coverage begins with a low surface density in Si-O- by flushing 0.01 M HCl followed by 0.5 ppm PEO to suppress the electrodispersion at the sampling junction and to delay the fast moving components. Back flushing the channel with 0.1 M NaOH from the end to enrich the surface in silanol group speeds up the late emergent peaks. The labor of repeating steps gets help from a homemade programmable Hi-voltage divider.

The gradient surface in silanolate density so prepared excels all other tries. Not only does it improve the sensitivity of the late emergent peaks by 30%, R2 also improves from 0.990 to 0.995.
摘要 I
英文摘要 III
圖目錄 VIII
表目錄 XII
第一章 緒論 13
1-1 研究背景 13
1-2 微晶片電泳之發展 15
1-2.1 玻璃基材 16
1-2.2 石英基材 17
1-2.3 高分子聚合物基材 17
1-3 電泳技術的發展及原理 18
1-3.1 電泳原理 21
1-3.2 毛細管電泳基本分離模式 26
1-4 微晶片電泳的偵測方式 28
1-4.1 光學偵測法 28
1-4.2 化學發光偵測法 29
1-4.3 電化學偵測法 30
1-5 接觸角 35
1-5.1 物理意義 36
1-5.2 靜態接觸角 37
1-5.3 毛細管電泳的管柱修飾 38
第二章 實驗部份 41
2-1 實驗架構 41
2-2 試劑 42
2-3 電泳晶片與相關儀器設備 44
2-3.1 晶片製程相關儀器 44
2-3.2 微電泳晶片實驗相關儀器 44
2-4 微電泳晶片製作流程 47
2-4.1 玻璃基材清洗 47
2-4.2 蒸鍍金屬阻障層 47
2-4.3 光阻塗佈與軟烤 48
2-4.4 曝光顯影與硬烤 48
2-4.5 化學濕式蝕刻 49
2-4.6 放電加工穿孔 51
2-4.7 高溫熔融接合 52
2-5 微晶片電泳實驗 53
2-5.1 微晶片電泳之基本操作模式 53
2-5.2 鉑電極外接式晶片之製作 54
2-5.3 鉑電極外接式晶片之效能檢測 55
2-5.4 實驗後微晶片電泳之清洗 56
2-6 微電泳晶片結合時序控制器自動分析模式的建立 56
2-6.1 微電泳晶片結合時序控制器 56
2-6.2 自動分析模式的建立 57
第三章 結果與討論 60
3-1 先導實驗的結果與討論 60
3-1.1 接觸角的量測 60
3-2 微電泳晶片結合時序控制器自動分析模式的建立 62
3-2.1 晶片效能的測試 62
3-2.2 自動分析模式的建立 64
3-3 偵測條件的最佳化 68
3-3.1 循環伏安法測試的探討 68
3-3.2 流體動力伏安法 (Hydrodynamic Voltammetry, HDV) 72
3-3.3 分離電壓與進樣時間對樣品分離效果的探討 73
3-3.4 系統的再現性與檢量線的建立 75
3-4 修飾流道 76
3-4.1 酸修飾進樣流道 76
3-4.2 聚環氧乙烷濃度的最佳化 83
3-4.3 雙向修飾聚環氧乙烷於進樣流道條件之探討 90
3-4.4 二次修飾效應的測試 97
3-4.5 流道表面梯度修飾之建立 106
3-4.6 系統穩定性的確認及檢量線建立 110
第四章 結論 113
第五章 參考文獻 115
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