臺灣博碩士論文加值系統

腸病毒71型曾經在台灣爆發多次疫情，造成數以千計人感染，而此時新型冠狀病毒(coronavirus)大流行並影響全球公共衛生。由於在病毒早期診斷中，傳統的偵測方法如細胞培養和PCR核酸檢測法較耗時，腸病毒71型和新型冠狀病毒都具有高傳染性，且新冠病毒尚未研發出疫苗，因此在本研究中，我們開發針對腸病毒71型與新型冠狀病毒的快速高靈敏度電化學感測鈀電極。
我們的成果顯示，感測鈀電極可以在15分鐘內區分腸病毒71型和克沙奇A16(Coxsackievirus A16)，其偵測極限可達單分子層次；此外，新冠病毒感測探針能在30分鐘內偵測新型冠狀病毒受體結合區域棘蛋白質的接合，其偵測極限可達數百nM以下。我們建立電化學阻抗頻譜的病毒偵測平台能提供一個新型的腸病毒71型和新型冠狀病毒偵測方法。

Enterovirus 71 outbreaks frequently in Taiwan. Thousands of children have been infected yearly. Recently, COVID-19 pandemic is affecting global public health severely. These two infectious diseases are highly contagious and COVID-19 do not have vaccines yet. Conventional detection approaches, such as cell culture and RT-PCR processes are time-consuming those cannot be used on site. We have developed a functionalized Electrochemical impedance spectroscopy (EIS) sensing nano-thin film palladium probes that can detect viruses rapidly and sensitively. Our results showed that the functionalized probes can distinguish EV71 from Coxsackievirus A16 (CVA16) and the limit of detection is about single molecule level in 15 min. Furthermore, the other functionalized probes can bind SARS-CoV 2 spike protein receptor-binding domain in 30 min directly. The limit of detection is about sub micro molar. We established the EIS detection model that provide a potential method to diagnose EV71 and coronavirus.

摘要 i
致謝 iii
表目錄 viii
圖目錄 ix
第一章 緒論 1
1.1研究背景 1
1.1.1研究動機 1
1.1.2病毒 1
1.1.3感染途徑 3
1.1.4病毒的治療與預防 5
1.1.5病毒偵測 5
1.2本研究偵測的病毒-腸病毒 7
1.2.1腸病毒介紹 7
1.2.2感染途徑 8
1.2.3臨床症狀 8
1.2.4流行病學 9
1.2.5腸病毒偵測 10
1.3本研偵測的病毒-新型冠狀病毒 11
1.3.1新冠病毒介紹 11
1.3.2感染途徑 13
1.3.3病徵 13
1.3.4流行病學 14
1.3.5新冠病毒偵測 15
1.4生物感測器 16
1.4.1生物感測器的發展 16
1.4.2生物感測器的重要性 17
第二章 研究策略 19
2.1實驗設計 19
2.2電化學偵測流程 21
第三章 材料與方法 22
3.1實驗材料 22
3.1.1電化學溶液配置 23
3.2目標蛋白質製備 24
3.2.1 IPTG誘導蛋白質大量表現 24
3.2.2蛋白質重摺疊 24
3.3蛋白質物理特性分析 26
3.3.1動態光散射粒徑分析 26
3.3.2蛋白質表面電位分析 27
3.3.3圓二色光譜與蛋白質二級結構 27
3.4 蛋白質接合能力試驗 28
3.5 電化學感測 28
3.5.1 循環伏安法(Cyclic voltammetry, CV) 28
3.5.2 電化學阻抗頻譜 30
3.6實驗設備 32
3.6.1電化學測量系統 32
3.6.2電極製備 32
3.6.3工作電極原理與製備 33
3.6.4參考電極 33
3.6.5輔助電極 34
第四章 實驗結果 35
4.1 腸病毒接合蛋白開發 35
4.1.1腸病毒VP1之鍵結蛋白序列 35
4.1.2腸病毒外殼蛋白rVP1表現 35
4.1.3 rEVBP蛋白之表現 37
4.2 蛋白質物化特性分析 39
4.2.1 蛋白質粒徑大小分析 39
4.2.1.1 rVP1粒徑分析 39
4.2.1.2 rEVBP粒徑分析 40
4.2.2 表面電位檢測 40
4.2.3 二級結構分析 41
4.3 EVBP與VP1接合能力檢測 44
4.3.1 ELISA分析rEVBP與rVP1之接合 44
4.3.2 Dot blot分析rEVBP與rVP1之接合 45
4.4 腸病毒的電化學偵測 47
4.4.1 rEVBP與鈀電極的接合 47
4.4.2 rEVBP修飾電極偵測rVP1 49
4.4.3 rEVBP修飾電極偵測BSA、Lysozyme 51
4.4.4 rEVBP修飾電極偵測腸病毒71型 52
4.5 新冠病毒偵測電極開發的蛋白質表現與純化 54
4.5.1新型冠狀病毒受體結合區域棘蛋白的表現與純化 54
4.5.2新冠病毒受體ACE2的表現與純化 55
4.6 ACE2受體與spike RBD的二級結構分析 57
4.7 ACE2與spike RBD接合能力檢測 59
4.7.1 ELISA分析ACE2與spike之接合 59
4.7.2 Dot blot分析ACE2與spike RBD之接合 60
4.8 新冠病毒的電化學偵測 62
4.8.1 ACE2與鈀電極的接合 62
4.8.2 ACE2修飾電極偵測spike RBD 63
4.8.3 ACE2修飾電極偵測BSA、Lysozyme 65
第五章 討論 67
5.1鈀電極修飾 67
5.2病毒蛋白之代表性 67
5.3 腸病毒蛋白rVP1的聚集 68
5.4 電化學偵測腸病毒之可行性 68
5.5電化學偵測新冠病毒之可能性 70
第六章 結論 71
第七章 參考文獻 72
第八章 附錄 83

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