臺灣博碩士論文加值系統

台灣乃慢性腎臟病盛行率與發病率居最高的國家，在早期很難被發覺，導致病情惡化到末期，患者需要終身血液透析或腎移植來維持生命。目前臨床上常被使用尿液試紙易造成偽陰性問題。此外，較精確尿液檢測需到健檢中心及實驗室，不利於居家檢測。因此提供操作簡單方便、非侵入式及高靈敏性的檢測機制，便於患者家中即時檢測，以達到早期發現早期治療的目標。微量白蛋白尿（microalbuminuria）是目前臨床上能最早診斷出慢性腎臟病的重要指標。
本研究為開發具抗體電活性探針的電化學免疫感測晶片。利用1-乙基-3-(3-二甲基氨基丙基)碳醯二亞胺/N-羥基琥珀醯亞胺（EDC/NHS）將二茂羧酸上的羧酸根活化，使其與人類血清白蛋白(HSA)抗體上的胺基交聯形成二茂鐵（ferrocene）與抗體的複合物，可透過傅立葉轉換中紅外線光譜儀（FTIR）及紫外線-可見光光譜（UV-vis）等技術分析二茂鐵與HSA抗體的交聯狀況。接著測試金奈米晶體能均勻沉積於網印電極表面上的最佳參數條件，使其能讓固定在電極上抗體量增加以提高靈敏度，可透過掃描式電子顯微鏡觀察電極表面的修飾狀況。最後將交聯二茂鐵的HSA抗體同時當作捕獲和訊號抗體，固定在沉積金奈米晶體後的電極上，完成晶片的備製。當免疫反應後，直接檢測二茂鐵的氧化電流訊號，以此推估HSA蛋白的濃度，經微分脈衝伏安法（differential pulse voltammetry，DPV）進行測量後，其微量白蛋白檢測濃度範圍為3-300 μg/mL，檢測極限為3 μg/mL。結果顯示本研究所開發之具抗體電活性探針的電化學免疫感測晶片針對為微量白蛋白尿具有良好的感測性能。

Chronic kidney disease (CKD) is recognized as a global health problem. Taiwan is the country with the highest incidence and prevalence rates of end stage renal disease. CKD is difficult to be diagnosed at an early stage which results in ineffective treatment and a need to rely on hemodialysis or kidney transplantation. Calorimetric urinary dipsticks are often used clinically which are semi-quantitative and can often result in inaccurate diagnostics. In addition, more accurate urine testing requires a well-equipped health examination center and laboratory which is not conducive to home testing. Therefore, we aim to develop a simple, user-friendly, non-invasive, quantitative and sensitive immunosensor which is convenient for point of care testing to achieve the goal of early detection and improved treatment. Microalbuminuria is currently the most widely used clinically viable indicator for early detection of chronic kidney disease.
In this study, we report the development of antibody-electroactive probe conjugate based electrochemical immunosensor for detection of microalbuminuria using differential pulse voltammetry (DPV). We use 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride/N-hydroxy succinimide (EDC/NHS) cross-linker to activate the carboxylate on the ferrocenecarboxylic acid to cross-link it with the amine group on the human serum albumin (HSA) antibody to form the ferrocene/antibody complex. The cross-linking efficacy of ferrocene and HSA antibody was analyzed by Fourier transform mid-infrared spectrometer and ultraviolet-visible light spectroscopy. Next, we have optimized the electrodeposition parameters to ensure uniform gold nanoparticle modification on the screen-printed electrode surface which results in improved antibody immobilization and resulting sensitivity. The gold nanoparticle modified working electrode surface was characterized using a field emission scanning electron microscope. After electrodeposition of gold nanoparticles, the complex of ferrocene and HSA antibody was drop-casted on the electrode surface. Following immunoreaction, the oxidation peak current was directly detected in spiked PBS to estimate the concentration of HSA protein in the clinically relevant range of 3-300 μg/mL with a limit of detection of 3 μg/mL. The results show that the proposed electrochemical immunosensor utilizing antibody-electroactive probe shows potential for point of care testing of CKD and can be used for improved public healthcare monitoring.

論文審定書 i
摘要 ii
Abstract iii
目錄 v
圖次 vii
表次 x
第一章 緒論 1
1.1 前言 1
1.2 研究背景 2
1.2.1 慢性腎臟病 2
1.2.2 目前慢性腎臟病檢測的方法 5
1.2.3 臨床問題與研究需求 8
1.3 研究目的 9
第二章 文獻回顧 10
2.1 應用於檢測人血清白蛋白的生物感測器 11
2.1.1 質量檢測方法（quality detection method） 12
2.1.2 電化學法（electrochemical method） 13
2.1.3 光學方法（optical method） 15
2.2 電活性標記交聯物的電化學免疫感測器應用 17
2.2.1 抗體與電活性探針的交聯 19
2.2.2 基於抗體-電活性探針交聯物的夾心免疫感測器 20
2.2.3 基於抗體-電活性探針交聯物的直接訊號免疫感測器 21
2.2.4 基於多抗體-電活性探針交聯物的免疫感測器 22
2.3 電化學分析法 23
2.3.1 電化學阻抗頻譜（electrochemical impedance spectroscopy, EIS） 25
2.3.2 微分脈衝伏安法（differential pulse voltammetry, DPV） 28
第三章 材料與方法 30
3.1 實驗耗材 30
3.2 實驗設備 31
3.3 網版印刷碳電極（screen-printing carbon electrode） 31
3.4 抗體嫁接於二茂鐵 32
3.5 電極表面修飾 33
3.6 免疫晶片分析 34
第四章 結果與討論 35
4.1 不同電化學沉積時間對於金奈米晶體之影響 35
4.2 二茂鐵與HSA抗體的交聯結果分析 37
4.3 有無具有二茂鐵交聯的HSA抗體之影響 39
4.4 不同白蛋白濃度下免疫分析之結果 40
第五章 結論與未來展望 44
參考文獻 46

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