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研究生： 林鑫
Lin, Shin
論文名稱： 電流式生物分子感測技術發展
Development of Amperometric Sensing Techniques for Biomolecules
指導教授： 周澤川
Chou, Tse-Chuan
學位類別： 博士
Doctor
系所名稱： 工學院 - 化學工程學系
Department of Chemical Engineering
論文出版年： 2005
畢業學年度： 93
語文別： 中文
論文頁數： 149
中文關鍵詞： C反應蛋白 、乙醯膽鹼 、感測器 、電流式 、掃描式電化學顯微鏡
外文關鍵詞： amperometry, acetylcholine, C reactive protein, scanning electrochemical microscopy
相關次數： 點閱：64  下載：3 
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    • 　　本研究以發展電流式感測技術為主，所選的生物分子分別是神經傳導物質-乙醯膽鹼與急性感染指標C反應蛋白。感測的系統依據感測物分為均相系統將感測物均勻分散在溶液中，與非均相感測系統利用抗體抗原間的選擇吸附將分析物固定在晶片上，之後接上過氧化酵素（horse radish peroxidase）進行感測。

    　　乙醯膽鹼的感測以鎳及電沈積鎳的催化系統Ni(OH)2/NiOOH，進行催化反應同時測量電流。電沈積鎳的表面用掃描式電子顯微鏡與原子力顯微鏡分析，在較高還原電流密度20 mA/cm2所得電極表面粗糙度較小。電流應答與乙醯膽鹼濃度依據極限電流反應式呈現性關係，在最適分析條件下鎳絲的應答電流(y)與乙醯膽鹼濃度(x)校正曲線為y=0.095x+0.196，而電沈積鎳為y=0.187x-1.476。

    　　C反應蛋白的分析，則是利用免疫吸附將C反應蛋白固定在分析晶片上，再用酵素連結產生電流訊號。同時針對掃描式電化學顯微鏡的系統作動力分析，分析參數包含C反應蛋白濃度、探針施加電壓與電探針面積。C反應蛋白的吸附動力分析中，在60分鐘後達到吸附平衡。掃描式電化學顯微鏡系統與免疫分析系統所結合的分析技術對C反應蛋白的感測濃度極限達0.1 µg/mL，在0.1～100 µg/mL的感測範圍有良好的線性關係，超過100 µg/mL則達到晶片的吸附飽和。

    　The amperometric techniques for sensing the bio-molecules including the neurotransmitter, acetylcholine, and the marker of the inflammatory response, C-reactive protein (CRP), were developed. The sensing systems were including the homogeneous phase that the analyte was dissolved in the solution, and the heterogeneous phase that the analyte was adsorbed selectively on the chip, binding with the horse radish peroxidase for scanning electrochemical microscopy (SECM) sensing.

    　The Ni(OH)2/NiOOH catalyst system was generated from Nickel wire and the electrochemical deposition Ni for sensing acetylcholine. The surface analysis of the Ni modified electrode was carried out by SEM and atomic force microscopy. The Ni deposited surface by high reduction current density resulted in the smoother morphology and better sensing performance for acetylcholine. The relation ship between the response current (y) and acetylcholine concentrations (x) was linearity according to limiting current equation. The sensing performance in the optima conditions obtained the calibration curves for Ni wire, y=0.095x+0.196, and for Ni modified electrode, y=0.187x-1.476.

    　The assay for C-reactive protein was using antibody to fix C-reactive protein on the chip, binding the enzyme to generate the current response. The kinetic analysis of the SECM system contented the variables including the CRP concentration, tip applied potential, and tip sensing area. The CRP adsorption kinetic was analyzed by SECM, and the time to adsorption equilibrium was 60 minutes. The CRP detection limit was 0.1 µg/mL by using SECM-ELISA system. The relation ship between response current and the CRP concentrations was good linearity in the 0.1～100 µg/mL CRP concentration region. The chip was approached the adsorption saturated when the CRP concentration was higher than 100 µg/mL.

    目錄 中文摘要 Ⅰ 英文摘要 Ⅱ 致謝 Ⅳ 目錄 Ⅵ 表目錄 Ⅶ 圖目錄 Ⅶ 符號說明 IV 第一章 簡介 1 1.1 生物分子簡介 1 1.1.1 神經傳導物質-乙醯膽鹼 1 1.1.2 急性感染指標-C反應蛋白 4 1.2 論文回顧 8 1.2.1 乙醯膽鹼感測器發展 8 1.2.2 C-反應蛋白分析方法 13 1.3 掃瞄式電化學顯微鏡簡介 14 1.3.1 掃瞄式電化學顯微鏡原理 14 1.3.2 掃瞄式電化學顯微鏡探針發展 20 1.3.3 掃瞄式電化學顯微鏡應用 27 1.3.4 生化系統分析 30 1.4 研究動機 35 第二章 電化學分析原理 36 2.1 乙醯膽鹼在鎳電極的電化學反應機構 36 2.1.1 乙醯膽鹼在鎳電極的電化學反應機構與反應動力 39 2.1.2 乙醯膽鹼在鎳電極的質傳模式 41 2.2 電化學CRP免疫分析原理 45 第三章 實驗設備與步驟 51 3.1 實驗儀器與藥品 51 3.1.1儀器設備 51 3.1.2實驗藥品 52 3.2 實驗步驟 53 3.2.1 乙醯膽鹼電化學感測 53 3.2.1.1 乙醯膽鹼的電化學行為 53 3.2.1.2 鎳電極對乙醯膽鹼的感測 54 3.2.1.3 電沈積鎳的製備與表面分析 54 3.2.1.4 電沈積鎳的電化學行為與對乙醯膽鹼的感測 55 3.2.2 CRP電化學免疫分析 56 3.2.2.1 CRP免疫晶片製作 56 3.2.2.2 電化學掃瞄與偵測CRP免疫感測晶片 59 3.2.2.2.1 掃瞄式電化學系統建立 61 3.2.2.2.2 電化學反應動力式探討 63 3.2.2.2.3 CRP吸附動力分析與SECM感測 67 第四章 結果與討論 71 4.1 乙醯膽鹼電化學感測 71 4.1.1 鎳電極與電沈積鎳的電化學行為 71 4.1.2乙醯膽鹼對鎳絲的電位窗 75 4.1.3 鎳電極製備條件對乙醯膽鹼的感測影響 83 4.1.3.1 溫度的影響 83 4.1.3.2 還原電流的影響與電極表面分析 85 4.1.3.3電解液pH值與攪拌速率對ACh感測影響 91 4.1.4 乙醯膽鹼的感測 95 4.1.5 其他種類神經傳導物質活性測試 101 4.2 CRP免疫晶片電化學分析 104 4.2.1 CRP免疫晶片的電化學特性 104 4.2.2 SECM對CRP電化學動力分析 110 4.2.3 CRP 二次抗體非特異性吸附分析 117 4.2.4 CRP免疫晶片吸附動力分析 120 4.2.5 CRP免疫晶片選擇性分析 123 4.2.6 CRP濃度在免疫晶片上之分析 125 4.3 綜合討論 129 4.3.1 乙醯膽鹼在鎳電極表面反應的擴散係數與擴散層厚度 129 4.3.2 CRP理論逼近曲線 133 第五章 結論與未來發展方向 136 5.1 結論 136 5.2 未來發展方向 137 第六章 參考文獻 138 附錄A 144 A-1 白金超微小電極製備與封裝 144 A-2 白金超微小電極電化學測試 146 個人著作 149 自述149

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