具生物沾粘性免疫奈米磁珠之電化學平台於急性冠心病標誌物檢測

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姓名

周忠佑(Chung-Yu Chou) 查詢紙本館藏

畢業系所

生醫科學與工程學系

論文名稱

具生物沾粘性免疫奈米磁珠之電化學平台於急性冠心病標誌物檢測
(Anti-biofouling Immuno-nanomagnetic Beads Integrate Electrochemistry Platform for Biomarker of Acute Coronary Heart Disease Detection)

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摘要(中)

電化學檢測系統在生物醫學中有著非常重要之地位，相較於傳統光學檢測方法，藉由電化學檢測系統即可快速得到高靈敏之檢測數據，但是目前電化學生物檢測平台因生物檢體的複雜性，造成檢測時非特異性蛋白貼附而導致偽陽性與偽陰性的可能大幅地增加，進而影響檢測結果。
本研究中所提出之具生物抗沾黏免疫奈米磁珠之電化學平台，係藉由奈米磁珠高表面積之特性，提高抗體-抗原反應性，利用強磁場進行高度預濃縮，增加其靈敏度，並減少反應時間。同時透過在磁珠表面進行改質，修飾兩性雙離子自組裝層，於表面形成強大水合層降低非特異性蛋白貼附，減少偵測背景值雜訊。即使在模擬血清中進行檢測，偵測極限(LOD) 仍然可以維持在19 pg/ml，並降低偽陽性與偽陰性的可能性。同時透過3,3′,5,5′-Tetramethyl Benzidine (TMB) 與Horseradish peroxidase (HRP) 在電化學檢測平台之下之氧化還原反應，在2分鐘內即可得到檢測結果，且由於元件體積微小的特性，藉此實現POCT之理念。該系統能針對臨床醫療帶來全新的改革，尤其在醫療資源匱乏之地區，增加對疾病即時檢測的能力並實現早期治療。
本論文以具抗生物沾黏性免疫奈米磁珠之電化學平台針對急性冠心病標誌物心肌肌鈣蛋白I (cTnI)　進行實際分析，實驗分析結果證明即使使用模擬血清當作溶劑，仍然可以使用150ul樣品量在pg/ml 的臨床檢測區間成功檢驗出正確濃度，實現快速且高準確度cTnI檢測之目的，為現有的cTnI分析方法提供即時檢測，並有效滿足基層醫療機構的檢測需求。

摘要(英)

The electrochemistry detection system plays a very important role in the biomedical field. In comparison with the traditional bioassays method, the electrochemistry detection system provides a high sensitivity result rapidly. However, the complexity of biological samples which may increase the probability of false positive or false negative greatly due to the non-specific protein adsorption will affect the result.
In this research, we utilized the characteristic of nanomagnetic beads’ high surface area to enhance antigen-antibody reaction and strong magnetic field for preconcentration to increase the sensitivity and decrease the operation time. At the same time, the surface of the beads was modified with zwitter ion self-assembly monolayer to form a hydration shell for reducing non-specific protein adsorption and background interference. Even the simulated serum was used as the biological sample, the limit of detection still maintained at 19 pg/ml and reduced the probability of false positive or false negative. Simultaneously, through the reduction-oxidation reaction between 3,3′,5,5′-Tetramethyl Benzidine (TMB) and Horseradish peroxidase (HRP) under the electrochemistry detection platform, we can get the result in two minutes. In addition, the component is small enough to realize the concept of POCT. The system we developed will bring innovation in clinical medicine, especially in areas where the medical resources are scarce, and increase the ability of real-time detection and early treatment.
In this research, anti-biofouling immuno-nanomagnetic beads integrated with an electrochemistry platform were used to detect biomarker of acute coronary heart disease, cardiac troponin I to verify the system. The results proved the system can use samples of 150 ul to detect the correct concentration from pg/ml to ng/ml in simulated serum successfully. The system achieved the goal of fast and high accuracy of cTnI detection which provides real-time detection and fulfills the detection needs for primary care.

關鍵字(中)

★ 電化學
★ 抗生物沾黏
★ 心肌梗塞
★ 心肌鈣鈣蛋白 I
★ 及時檢驗

關鍵字(英)

★ Electrochemistry
★ anti-biofouling,
★ Myocardial infarction (MI)
★ cardiac troponin I (cTnI)
★ real-time detection

論文目次

目錄
中文摘要 I
Abstract II
誌謝 IV
目錄 V
圖目錄 VII
表目錄 IX
專有名詞縮寫對照表 X
符號說明 XI
第一章緒論 1
1-1 前言 1
1-2 研究動機和目的 2
1-3 文獻回顧 3
1-3-1 心肌梗塞 3
1-3-1心電圖 6
1-3-2生物標誌物 8
1-4 論文架構 10
1-5 系統架構 11
1-6 藥品與儀器 12
第二章奈米磁珠 14
2-1背景 14
2-2奈米磁珠合成 15
2-2-1磁核合成流程 20
2-2-2矽層生成 21
2-2-3自組裝單層膜修飾 22
2-3 奈米磁珠粒徑分析 23
2-3-1背景 23
2-3-2 穿透式電子顯微鏡樣品製備 23
2-3-3磁珠粒徑分析結果 23
2-4 奈米磁珠XPS 分析 25
2-4-1背景與原理 25
2-4-2 XPS樣品製備 25
2-4-3 奈米磁珠XPS結果與分析 26
2-5 奈米磁珠表面蛋白質定量分析 28
2-5-1背景與原理 28
2-5-2 BSA樣品製備與量測 31
2-5-3 磁珠共軛 BSA樣品製備與量測 32
2-5-4 磁珠表面蛋白質分析結果 33
第三章生物分子固定 35
3-1 背景與原理 35
3-2 樣品製備 37
第四章電化學系統與ELISA於cTnI之檢測 40
4-1 背景與原理 40
4-2 電化學系統檢測操作流程 44
4-3 電化學檢測cTnI濃度校正曲線 45
4-4 於擬真血清中cTnI之檢測 46
4-5 於擬真血漿中cTnI之檢測 47
4-6 ELISA cTnI濃度校正曲線 48
4-6-1 背景與原理 48
4-6-2 ELISA檢測操作流程 48
4-6-3 ELISA檢測分析結果 50
4-7 二方法之結果曲線比較 51
4-8 操作時間比較分析 52
第五章結論 55
第六章未來展望 56
參考文獻 57

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指導教授

黃貞翰(Chen-Han Huang)

審核日期

2020-6-24

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