於矽奈米線場效應電晶體利用核酸適體 三明治法檢測心肌肌鈣蛋白I之研究

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姓名

楊文旺(Wen-Wang Yang) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

於矽奈米線場效應電晶體利用核酸適體三明治法檢測心肌肌鈣蛋白I之研究
(Aptamer Sandwich Assay for an Ultra-high Sensitivity Detection of Cardiac Troponin I by Silicon Nanowire Field-effect Transistor)

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至系統瀏覽論文 (2025-8-1以後開放)

摘要(中)

急性心肌梗塞 (Acute myocardial infarction, AMI) 為全球心血管相關疾病的主要死亡原因，當發生心肌梗塞時，心肌會釋放不同心臟生物標誌物，其中心肌肌鈣蛋白I (cardiac troponin I, cTnI) 對於急性心肌梗塞具有極高專一性，因此被視為檢測急性心肌梗塞的黃金標準。
目前多數實驗室使用歐洲心臟協會(European Society of Cardiology, ESC) 的0/1-h algorithm作為急性心肌梗塞早期診斷的方式，依據在第0小時血液中的cTnI濃度與1小時後的濃度變化對病患進行分類，因此檢測平台的靈敏度與解析度是診斷的一大重點。此外，急性心肌梗塞與心臟病發及心臟驟停密切相關，若能在抵達急診室前進行檢測，有助於提早預判病患之病程，降低死亡率。為了實現上述目標，發展高靈敏性與解析度的定點照護 (Point of care, POC) 設備是必要的。
矽奈米線場效應電晶體 (Silicon nanowire field effect transistors, SiNW-FETs) 因其具有高靈敏性、即時檢測、免標定與易攜帶等優點，被視為極具發展潛力檢測平台。此外，使用兩種對於cTnI有著不同結合位點的核酸適體 (Aptamer, Tro4 & Tro6) 做為檢測探針，透過三明治法 (Sandwich assay) 進行檢測，不僅增加檢測專一性，也有放大檢測訊號的可能性。
本研究使用Mixed-SAMs (silane-PEG-NH2：silane-PEG-OH = 1：
10 (mM/mM)) 與戊二醛 (Glutaraldehyde) 對SiNWFET表面進行表面改質，完成Tro4的表面固定化。接著，透過原子力顯微鏡以及化學分析線光子能譜儀，進行表面改質確認。我們也使用COB (Chip on board) 系統於不同鹽離子濃度的環境下檢測cTnI，確認最佳檢測環境。最後，藉由COB系統確認加入Tro6對於cTnI檢測之影響。
由研究結果得知，使用核酸適體三明治 (Aptamer sandwich assay)，並在高鹽離子濃度 (1xPBS) 下進行檢測，加入Tro6的電訊號變化量與未加入前相比有著顯著的提升。此外，在高鹽離子濃度下可以維持核酸適體與cTnI的結合穩定性，因此cTnI與電訊號有著更高的相關性，且解析度更大。在最佳化條件下檢測臨床檢體中的cTnI濃度，透過1xPBS與血漿樣品稀釋液進行稀釋，分別得到LOD為0.151 pg/mL及0.861 pg/mL，解析度為0.012 pg/mL與0.525 pg/mL，低於許多臨床檢測所需，證實本研究製備的cTnI生物感測器在臨床應用上具有很高的診斷潛力。

摘要(英)

Rapid diagnosis and treatment for acute myocardial infarction (AMI), which is closely related to heart attacks and cardiac arrests and is the leading cause of death from cardiovascular diseases worldwide, is essential to save patient’s life. Among the cardiac biomarkers which are released from the cardiac muscle when AMI occurs, cardiac troponin I (cTnI) is regarded as the “gold standard” for AMI diagnosis because of its high specificity and sensitivity to this disease. Currently, most laboratories use the 0/1-h algorithm of the European Society of Cardiology (ESC) as the method for early diagnosis of AMI. This approach combines a very low cTnI at the initial emergency department (ED) presentation and dynamic change of cTnI values between 0 and 1 hour to triage patients into rule-out, observation, and rule-in categories. To achieve the above objectives, ultrasensitive and high-resolution point of care test (POCT) equipment must be developed.
The silicon nanowire field effect transistors (SiNWFETs) were used as the detection platform because of have many advantages, such as high sensitivity, label-free detection, and real-time detection. In addition, we used two aptamers (Tro4 & Tro6) that have different binding sites for cTnI, and detect cTnI via sandwich detection. This detection technique not only has high specificity but also amplifies the signal.
In this study, we fabricated the biosensor by modifying the SiNWFETs with Mixed-SAMs (silane-PEG-NH2：silane-PEG-OH =1：10 (mM/mM)) and Glutaraldehyde to immobilize Tro4 aptamer and detect cTnI. The surface modification is confirmed by Atomic force microscopy (AFM) and Electron Spectroscopy for Chemical Analysis (ESCA) whereas the cTnI was detected in different salt concentrations by the COB (Chip on board) system to determine the optimal detection condition. Finally, the effect of adding the signal probe (aptamer, Tro6) on cTnI detection was confirmed by the COB system.
Based on the results of the study, the correlation between cTnI and electrical signals was found to be high when the Aptamer sandwich assay and high salt ion concentration (1xPBS) detection methods were used. Because the sandwich assay improves detection specificity, and at high salt concentrations, it maintains binding stability for aptamer and cTnI. Finally, under optimal conditions, the concentration of cTnI in clinical specimens was detected using a dilution of 1xPBS and plasma sample diluent. The limits of detection (LOD) obtained were 0.151 pg/mL and 0.861 pg/mL, with resolutions of 0.01 pg/mL and 0.53 pg/mL, respectively. These results are lower than the requirements for clinical assays, proving our device has a good potential to be used in the clinical assay.

關鍵字(中)

★ 矽奈米線場效電晶體
★ 生物感測器
★ 核酸適體三明治法
★ 心肌肌鈣蛋白I檢測

關鍵字(英)

論文目次

摘要 I
Abstract III
致謝 V
目錄 VII
圖目錄 X
表目錄 XIII
第一章緒論 1
第二章文獻回顧 3
2.1 核酸適體介紹 3
2.1.1 核酸適體 3
2.1.2 Systematic Evolution of Ligands by Exponential Enrichment 4
2.1.3 核酸適體與抗體之比較 6
2.1.4 核酸適體於場效應電晶體之應用 8
2.2 急性心肌梗塞介紹 11
2.2.1 急性心肌梗塞 (Acute myocardial infarction, AMI) 11
2.2.2 心臟生物標誌物 14
2.2.3 心肌肌鈣蛋白 (Cardiac troponin, cTn) 16
2.2.4 急性心肌梗塞之診斷 19
2.2.5 急性心肌梗塞診斷之瓶頸 22
2.3 心肌肌鈣蛋白I之檢測 23
2.3.1 cTnI之檢測 23
2.3.2 cTn檢測之市售設備 27
2.4 矽奈米線場效應電晶體 30
2.5 晶片表面改質 36
2.5.1 自組裝單層膜 36
2.5.2 表面分子固定化 39
2.5.3 聚乙二醇 (Polyethylene glycol, PEG) 於自組裝單層膜應用 41
第三章實驗藥品、儀器與方法 44
3.1 實驗架構 44
3.2 實驗藥品 46
3.3 儀器設備 48
3.4 緩衝液配置 49
3.5 實驗步驟 50
3.5.1 FETs表面改質 50
3.5.2 FETs電訊號量測 55
3.5.3 原子力顯微鏡(AFM)表面粗糙度分析 57
3.5.4 化學分析光電子能譜儀 (ESCA) 表面元素組成分析 57
3.5.5 Enzyme-linked immunosorbent assay (ELISA) 核酸適體-抗原結合穩定性測定 58
第四章結果與討論 60
4.1 表面改質之鑑定 60
4.1.1 原子力顯微鏡 (AFM) 60
4.1.2 化學分析光電子能譜儀 (ESCA) 63
4.2 不同鹽濃度對於檢測cTnI之影響 67
4.2.1 檢測環境鹽濃度對於Tro4與cTnI之結合穩定性 67
4.2.2 不同鹽濃度下之SiNWFETs 電訊號 69
4.3 利用核酸適體三明治法 (Aptamer sandwich assay) 進行cTnI檢測 73
4.4 cTnI於人類體液中之定量分析 78
4.5 cTnI臨床檢體之定量分析 80
第五章結論 82
第六章未來展望 84
第七章參考文獻 85

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指導教授

陳文逸(Wen-Yih Chen)

審核日期

2023-7-20

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