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研究生:陳冠中
研究生(外文):CHEN, GUAN-ZHONG
論文名稱:開發潛伏電化學氧化還原探針平台運用於比例定量捕獲活體細胞中之亮氨酸氨肽酶
論文名稱(外文):Development of Latent Electrochemical Redox Probe Platform for Ratiometric active profiling of Leucine Aminopeptidase in Living Sample
指導教授:黃聲東
指導教授(外文):HUANG, SHENG-TUNG
口試委員:郭憲壽汪昆立
口試委員(外文):KUO, HSIEN-SAWWANG, KUN-LI
口試日期:2019-07-02
學位類別:碩士
校院名稱:國立臺北科技大學
系所名稱:化學工程與生物科技系化學工程碩士班
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:58
中文關鍵詞:電化學亮氨酸氨肽酶酶生物傳感器肝臟疾病
外文關鍵詞:Electrochemical probeLeucine AminopeptidaseEnzymatic biosensor
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氨酸氨肽酶(LAP)是一種生物必需的蛋白酶,被認為有機會成為下一個肝臟惡性腫瘤的生物標誌物,其過度的表達造成致命生理和病理障礙有直接的關聯。因此我們設計了一種創新的電化學底物亮氨酸-氨基二茂鐵(Lap-FcP),能直接分析細胞中的LAP活性及選擇性。LAP迅速水解Lap-FcP的Leu殘基,通過設計潛伏式自我斷裂機制經由電子共振轉出氨基二茂鐵(AFc)。探針LAP-FcP對LAP具有高特異性並且沒有受其他電化學活性生物分子干擾。探針Lap-FcP賦予電化學傳感器強大的動態範圍和令人矚目的檢測極限2.14ng/ml。除此之外, Lap-FcP用於追踪活細胞(HepG2)表面及內部LAP活性,並透過抑制劑bestatin驗證確認。Lap-FcP電化學生物傳感器顯示出在監測HepG2細胞中的LAP活性表現出色可靠性。
Leucine aminopeptidase (LAP) is an essential proteolytic enzyme, and potential biomarker for liver malignancy; overexpression of LAP is directly linked with some fatal physiological, and pathological disorders. In this regard, we designed an innovative electrochemical substrate leucine-benzyl ferrocene carbamate (LAP-FcP) for direct activity based selective profiling of LAP activity in live cells. In practice, LAP instantaneously hydrolyze the Leu residue of the electrochemical substrate LAP-FC to eliminate the masked electrochemical reporter amino ferrocene (AF) via predefined self-immolative cascade. The probe LAP-FcP is very specific for LAP, and free of other electroactive biological interference. The probe LAP-FcP empowered electrochemical sensor displayed broad dynamic range and admirable detection limits2.14ng/ml. On top of this, the electrochemical substrate LAP-FcP was employed in real time tracking of endogenous LAP activity in live cells (Hep G2) on supplemented with LAP inhibitor bestatin. The Lap-FC substrate based electrochemical sensor showcased an excellent reliability towards monitoring cellular LAP activity in Hep G2
摘要 i
Abstract ii
致謝 iii
目錄 iv
圖目錄 vii
表目錄 ix
附圖目錄 x
第一章 前言 1
第二章文獻回顧 2
2.1即时檢测(Point-of-Care Testing) 2
2.2生物分子探針 3
2.2.1生物分子探針 3
2.2.2隱藏式自我斷裂 4
2.3亮氨酸氨肽酶(Leucine aminopeptidase) 5
2.4亮氨酸氨肽酶生理表現 7
2.4.1植物 7
2.4.2動物 7
2.4.2.1肝臟膽囊疾病 8
2.4.2.2懷孕疾病 8
2.4.2.3白內障疾病 9
2.5檢測平台 9
2.6電化學法 10
2.6.1電化學法原理及優勢 10
2.6.2循環伏安法 11
2.6.3微分脈衝伏安法 13
2.6.4 實驗裝置 14
第三章研究目的與策略 15
3.1實驗研究目的 15
3.2實驗研究策略 15
3.2.1探針設計概念 15
3.2.2電化學隱藏式探針LAPFcP 16
第四章 實驗方法與討論 17
4.1實驗藥品 17
4.1.1合成之藥品 17
4.1.2合成之溶劑 17
4.1.3緩衝溶液之藥品 18
4.1.4.干擾物之藥品 18
4.1.4真實樣品 19
4.2實驗儀器 19
4.2.1實驗使用之儀器 19
4.3探針合成設計 20
4.3.1 Compound(2)合成方法 20
4.3.2 Ferrocene Azide合成方法 21
4.3.3 LAP- Fc (3)合成方法 21
4.3.4 LAP- FcP(4)合成方法 22
4.4樣品配置 24
4.4.1緩衝溶液配置 24
4.4.2 LAP-FcP、Leucine Aminopeptidase儲備溶液 24
4.4.3電化學分析CV及DPV機制驗證 24
4.4.3.1電極清洗 24
4.4.4電化學分析線性檢量線 25
4.4.5電化學分析酵素動力學 25
4.4.6電化學分析干擾物 25
4.4.7電化學分析抑制劑Bestatin 25
4.4.8高效液相層析儀 26
4.4.9 真實樣品HepG2細胞 26
第五章 結果與討論 27
5.1 LAPFcP合成與設計機制 27
5.2 LAPFcP電化學探針機制驗證 28
5.2.1循環伏安法Cyclic voltammetry(CV)驗證 28
5.2.2微分脈衝伏安法Differential pulse voltammetry (DPV)驗證 29
5.2.3抑制劑Bestatin驗證 31
5.3檢測之最佳化參數 32
5.3.1最佳化參數-ph值 32
5.3.2檢測極限 33
5.3.3酵素動力學 34
5.3.4特異性測試 36
5.3.5真實樣品-細胞測試 38
第六章 結論 41
參考文獻 42
附錄 47

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