臺灣博碩士論文加值系統

肌酸酐為肌酸 (creatine) 與磷酸肌酸 (creatine phosphate) 之代謝廢物，為人體血液與尿液中作為評估腎臟功能的重要指標之一，臨床上常使用傑夫呈色法 (Jaffé reaction)，利用苦味酸 (picric acid) 與肌酸酐之呈色反應進行檢測。本研究期望以電化學方法結合傑夫呈色法以達到更快速且便利之定量手段。以3,5-二硝基苯甲酸 (3,5-dinitrobenzoic acid, DNB) 取代苦味酸，透過分光光度計、螢光光度計和紅外線光譜儀分析DNB與肌酸酐之呈色反應，本研究簡稱為類傑夫呈色法 (modified Jaffé reaction)。
本研究擬將DNB固定於以poly(Pyr-P3CA) 高分子膜修飾的工作電極上，poly(Pyr-P3CA) 是以吡咯 (pyrrole, Pyr) 與吡咯-3-羧酸 (pyrrole-3-carboxylic acid, P3CA) 經由電聚合方式形成的共聚膜。文中除了以分光光度計、螢光光度計和紅外線光譜儀分析確認DNB與肌酸酐之反應性，乃是以電化學電位式進行肌酸酐濃度的校正與檢測。此外不同的電極製備參數亦將於本研究中進行探討，同時，亦以本研究開發的肌酸酐感測器對尿液檢體進行感測與比對，以確認開發的感測器在臨床應用的可行性。

Creatinine, a metabolite from creatine and creatine phosphate, is a key indicator for kidney’s function in human’s bloodstream and urine. In clinical, Jaffé reaction is the most common method to detect creatinine, where picric acid can react with creatinine in alkaline condition. In this study, we use 3,5-dinitrobenzoic acid (DNB) instead of picric acid to achieve a more convenient and faster way in creatinine detection. By UV/Vis spectrophotometer, fluorescence spectrometer and FTIR, we can analyze the reaction between DNB and creatinine, named “modified Jaffé reaction”.
In this research, DNB is immobilized onto the working electrode by poly(Pyr-P3CA) film, where pyrrole (Pyr) and pyrrole-3-carboxylic acid (P3CA) are copolymerized by electropolymerization. We not only discuss the reactivity by UV/Vis spectrometer, fluorescence spectrometer and FTIR, but also optimize creatinine calibration and detection by potentiometry. Last but not least, the fabricated creatinine sensor will apply in urine spicemen detection to ensure the feasibility in clinical application.

中文摘要 I
Extended Abstract Ⅱ
誌謝 Ⅴ
表目錄 X
圖目錄 XI
第一章 緒論 1
1-1前言 1
1-2腎功能 (Kidney’s function) 1
1-3研究動機及目的 2
第二章 文獻回顧 3
2-1 肌酸酐 (Creatinine) 3
2-1-1 簡介 3
2-1-2 臨床評估 3
2-2 生醫感測器概述 4
2-2-1 發展 4
2-2-2 生物識別元件 (Bioreceptor) 5
2-2-3 識別元件的固定化 9
2-2-4 超分子化學識別法 11
2-2-5 生物抗汙 (Antibiofouling) 12
2-2-6生醫感測器種類 12
2-3 肌酸酐感測技術 18
2-3-1 呈色法 (Colorimetry) 18
2-3-2 酵素法 (Enzymatic method) 19
2-3-3 電化學法 (Electrochemistry) 20
2-4 電化學方法 20
2-4-1 電化學感測途徑 20
2-4-2 線性掃描伏安法 (Linear sweep voltammetry, LSV) 20
2-4-3 循環伏安法 (Cyclic voltammetry, CV) 21
2-4-4 脈衝伏安法 (Pulse voltammetry, PV) 21
2-4-5 電化學阻抗分析 (Electrochemical impedance spectroscopy, EIS) 22
2-4-6 安培法 (Amperometry) 23
第三章 實驗方法、材料與儀 25
3-1 以HPLC 製備肌酸酐標準尿溶液之檢量線 25
3-2 以UV/Vis 分光光度計分析肌酸酐呈色反應 25
3-3 以螢光光譜儀分析肌酸酐呈色反應 25
3-3-1 肌酸酐呈色反應圖譜 25
3-3-2 Job plot分析 26
3-4 以FTIR 分析肌酸酐呈色反應 26
3-5 工作電極之製備 26
3-5-1 裸金電極 (Bare Au) 26
3-5-2 Polypyrrole 修飾電極 26
3-5-3 PPy-DNB 修飾電極 26
3-5-4 Poly(Pyr-P3CA)-DNB 修飾電極 27
3-5-5 三極式電化學系統 27
3-6 以SEM 掃描式電子顯微鏡觀察電極表面 28
3-7 以修飾電極進行電活性探討 28
3-8 肌酸酐之感測條件最佳化 28
3-8-1 背景攪拌速率 28
3-8-2 背景NaOH 濃度 28
3-8-3 背景pH 29
3-8-4 前置平衡時間 29
3-9 修飾電極之電聚合條件最佳化 29
3-9-1 掃描速率 29
3-9-2 掃描圈數 29
3-9-3 DNB濃度及有無P3CA 29
3-9-4 電聚合pH 29
3-9-5 浸泡條件之選擇 30
3-10 稀釋肌酸酐標準尿溶液之檢量線 30
3-11 選擇性測試 30
3-11-1單針測試 31
3-11-2連續測試 31
3-12 臨床尿液檢體檢測 31
3-13 電極保存穩定性測試 31
3-14 相關儀器介紹 32
3-14-1 高效液相層析法 (High performance liquid chromatographer, HPLC) 32
3-14-2 傅立葉轉換紅外線光譜儀 (Fourier Transform Infrared Spectrometer, FTIR) 32
3-14-4 掃描式電子顯微鏡 (Scanning Electron Microscope, SEM) 32
3-15 實驗藥品 33
3-16 實驗儀器 34
第四章 結果與討論 35
4-1 肌酸酐反應之呈色分析 36
4-2 肌酸酐反應之螢光分析 40
4-2-1 以螢光光度計半定量分析肌酸酐反應 40
4-2-2 以Job plot探討類傑夫法產物 43
4-3 以FTIR分析類傑夫法反應機構 43
4-4 電化學系統分析 47
4-4-1 背景攪拌速率探討 47
4-4-2 背景NaOH 濃度探討 47
4-4-3 背景pH探討 49
4-4-4 前置平衡時間探討 50
4-5 電極修飾分析 52
4-5-1 SEM影像 52
4-5-2 電極電活性測試 53
4-5-3 電聚合掃描速率與圈數探討 54
4-5-4 電聚合反應物濃度探討 56
4-5-5 電聚合環境pH探討 58
4-5-6以浸泡法探討電極檢測之檢量線 60
4-6 稀釋倍率探討 63
4-7選擇性探討 66
4-8 臨床檢體檢測 67
4-9 電極穩定性測試 70
4-10 本研究與過去文獻比較 71
第五章 結論 73
參考文獻 74

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