磁電化學免疫分析系統 於新型冠狀病毒感染檢測之研製

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姓名

周怡萱(I-Hsuan Chou) 查詢紙本館藏

畢業系所

生醫科學與工程學系

論文名稱

磁電化學免疫分析系統於新型冠狀病毒感染檢測之研製
(Research and development of magneto-electrochemical immunoassay system for SARS-CoV-2 infection detection)

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摘要(中)

由嚴重急性呼吸系統綜合症冠狀病毒第二型(Severe Acute Respiratory Syndrome Coronavirus 2)引起的2019新冠肺炎(Coronavirus disease 2019)，其影響擴及全球，已對人們的生活型態造成改變。受感染者從輕微的類流感症狀，如發燒、咳嗽和味覺喪失，至嚴重的呼吸道疾病造成呼吸困難、休克甚至多重器官衰竭以致危及性命。致使此疫情難以掌控的最大問題，在於此病毒的高度傳染性以及潛伏期的無症狀感染。
目前常用於診斷COVID-19之核酸檢測方法較為耗時，需要專業人員的操作和儀器設備的要求，讓檢測侷限在特定的場域。而針對抗原的免疫檢測則有操作較為簡單快速的優點，更具便利性。因此，本篇研究目標為利用免疫磁珠結合電化學方法研發一項檢測快速且具可靠靈敏度的可攜式感測系統。利用特殊自組裝單層膜進行磁性奈米粒子之表面改質與修飾，製備具抗非特異性吸附之免疫磁珠以進行目標分子的捕捉和預濃縮，進而提高檢測之靈敏度與準確性。為方便使用者自行採檢，本研究的檢體預計選擇採用受測者之唾液，並針對病毒之核殼蛋白(Nucleocapsid protein)做為檢測目標物。我們利用自行配製之核殼蛋白標準樣品溶液制定系統之標準檢量線，並以含有核殼蛋白的人工唾液受試於本系統。本研究結果之檢測線性區間落於臨床診斷的濃度範圍，系統之檢測極限(LOD)可達1.12 ng/mL，故本方法有檢測快速之優點以及具可靠靈敏度，且有達到即時現場檢測之潛力與可行性。

摘要(英)

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has expanded globally and changed people’s way of life. The infected may have mild flu-like symptoms of fever, cough and ageusia to severe respiratory illness with dyspnoea, shock and multiple organ failure, which is life-threatening. The high infectiousness of the virus and asymptomatic infection during its incubation period make the epidemic hard to control. Thus, this study aims to develop a portable magneto-electrochemical immunoassay system that allows rapid detection and reliable sensitivity. We synthesized a magnetic nanobeads-based biomaterial with the self-assembled monolayer coated and coupling with selected antibodies. Using the virtue of immunomagnetic beads, the property can decrease non-specific binding, increase system sensitivity, and achieve pre-concentration through magnetically captured during the detection process. For easier sample self-collection to users without trained personnel, this research selects saliva as a specimen, focusing on nucleocapsid protein of the virus for detection target. We analyze the shape and size of the prepared magnetic beads by transmission electron microscope. Then we test our system by using a homemade sample of standard solution and artificial saliva with nucleocapsid protein. On the other hand, we also perform an enzyme-linked immunosorbent assay to compare and verify our approach. The results show that calibration curve′s linear interval falls within the clinical diagnosis′s concentration range, and the system’s detection limit achieves 1.12 ng/mL. Hence our system has the potential and possibility to accomplish real-time and on-site detection.

關鍵字(中)

★ 電化學
★ 奈米磁珠
★ 傳染性疾病
★ 新型冠狀病毒
★ 核殼蛋白
★ 即時現場檢測

關鍵字(英)

★ Electrochemistry
★ Magnetic nanobeads
★ Infectious disease
★ SARS-CoV-2
★ Nucleocapsid protein
★ Real-time and on-site detection

論文目次

中文摘要 i
Abstract ii
致謝 iii
目錄 iv
圖目錄 v
表目錄 v
第一章緒論(Introduction) 1
1-1 前言(Preface) 1
1-2 研究目的(Objectives) 1
1-3 文獻回顧(Literature review) 2
1-3-1 適應症(Indication) 2
1-3-2 檢測和診斷方法(Detection and Diagnostic methods) 3
1-3-3 生物標記物(Biomarker) 6
1-3-4 檢體前處理(Pretreatment of specimen) 7
1-4 論文架構(Framework of the thesis) 8
第二章材料與方法(Material and methods) 9
2-1 藥品與儀器(Chemicals and instruments) 9
2-2 功能性奈米磁珠(Functionalized magnetic nanobead) 10
2-2-1 磁珠製備(Preparation of MNB) 12
2-2-2 表面修飾(Surface modification) 14
2-2-3 特性分析(Characterization) 17
2-3 檢測樣品製備(Testing sample preparation) 20
2-3-1 標準N蛋白(Standard N protein sample) 20
2-3-2 人工唾液(Artificial saliva) 20
2-4 電化學系統(Electrochemistry system) 21
2-4-1 檢測原理(Fundamental) 21
2-4-2 實驗方法(Method) 25
2-5 傳統ELISA法(Traditional ELISA) 27
第三章實驗結果與討論(Results and Discussions) 29
3-1 標準曲線(Standard curve)/檢量線(Calibration curve) 29
3-1-1 電化學法(Electrochemistry) 29
3-1-2 傳統ELISA法(Traditional ELISA) 31
3-2 兩系統之比較(Comparation between the two methods) 32
3-3 人工唾液中N蛋白之檢測(Diagnostic of N protein in saliva) 36
第四章結論(Conclusions) 37
參考資料(References) 38

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指導教授

黃貞翰

審核日期

2021-9-8

推文