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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林致廷 | zh_TW |
| dc.contributor.advisor | Chih-Ting Lin | en |
| dc.contributor.author | 張凱俐 | zh_TW |
| dc.contributor.author | Kai-Li Chang | en |
| dc.date.accessioned | 2025-09-17T16:22:19Z | - |
| dc.date.available | 2025-09-18 | - |
| dc.date.copyright | 2025-09-17 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-08-07 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/99685 | - |
| dc.description.abstract | 本論文旨在建立一套結合 CRISPR/Cas 系統與以金為電極材料搭配 hairpin DNA 探針之電化學量測方法，並探討實驗中可能影響訊號表現的潛在干擾因子。研究中發現，當以電化學阻抗分析(Electrochemical Impedance Spectroscopy, EIS)量測 Cas12a 系統的剪切反應時，商用反應緩衝液中的重組白蛋白(recombinant albumin)可能造成非特異性吸附與介面阻擋效應，導致背景訊號上升，進而干擾目標訊號的辨識與解析。
為解決此問題，本研究調整量測流程，改以蛋白質預先覆蓋後的電極狀態作為新的基線條件，以降低蛋白質成分對 EIS 量測的干擾。此外，在使用排除蛋白質成分的緩衝液條件下進行剪切反應時，我們亦觀察到 hairpin DNA 探針的二級結構穩定性可能影響其作為 Cas12a 反式剪切底物的效果。因此，我們進一步參考相關文獻，整理過往 hairpin DNA 做為 Cas12a 反式剪切目標之設計，並歸納出在結構穩定性與反應效率間可進一步優化之方向。 綜合本研究結果，初步測試以hairpin DNA做為金電極表面探針之CRISPR/Cas12a 電化學生物感測方法，並針對其訊號準確性之干擾來源與潛在解決方案進行探討，期望能作為後續相關系統優化之參考依據。 | zh_TW |
| dc.description.abstract | This thesis aims to establish an electrochemical sensing platform that integrates the CRISPR/Cas system with gold electrodes modified by hairpin DNA probes, and to investigate potential interfering factors that may affect signal performance. During measurements of the Cas12a cleavage reaction using Electrochemical Impedance Spectroscopy (EIS), we found that recombinant albumin, commonly present in commercial reaction buffers, may lead to nonspecific adsorption and interfacial blocking effects. These effects result in elevated background signals, thereby interfering with the identification and interpretation of target signals.
To address this issue, we modified the measurement workflow by using the electrode surface pre-coated with proteins as a new baseline condition, which helped reduce the interference caused by protein components during EIS detection. In addition, under buffer conditions without albumin, we observed that the secondary structure stability of hairpin DNA probes could also influence their suitability as trans-cleavage substrates for Cas12a. Therefore, we further reviewed relevant literature and summarized prior designs of hairpin DNA used as Cas12a trans-cleavage targets, identifying possible directions for structural optimization to balance stability and reaction efficiency. In summary, this study provides preliminary testing of a CRISPR/Cas12a electrochemical biosensing approach based on hairpin DNA probes immobilized on gold electrodes, and discusses key sources of signal interference as well as potential solutions. The findings are expected to serve as a reference for future optimization of related sensing systems. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2025-09-17T16:22:19Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2025-09-17T16:22:19Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 口試委員審定書 i
致謝 iii 摘要 v Abstract vii 目次 ix 圖次 xiii 表次 xv 第一章 緒論 1 1.1 研究背景 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 生物感測技術及其發展 . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.3 電化學生物感測 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.4 基於 CRISPR/Cas 系統之電化學檢測 . . . . . . . . . . . . . . . . . . 5 1.5 研究動機 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 第二章 理論及實驗原理 9 2.1 電化學原理 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 2.1.1 法拉第與非法拉第過程 . . . . . . . . . . . . . . . . . . . . . . . 11 2.1.2 電雙層成因及其結構 . . . . . . . . . . . . . . . . . . . . . . . . 12 2.2 電化學量測 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 2.2.1 循環伏安法 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 2.2.2 電化學阻抗譜 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.3 CRISPR/Cas 技術 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 2.3.1 基因編輯技術發展 . . . . . . . . . . . . . . . . . . . . . . . . . . 23 2.3.2 CRISPR-Cas 系統的研究演進歷程 . . . . . . . . . . . . . . . . . 24 2.3.3 CRISPR-Cas 系統不同類型的原理與機制 . . . . . . . . . . . . . 25 2.4 DNA 探針表面封閉劑選擇 . . . . . . . . . . . . . . . . . . . . . . . 26 第三章 實驗架設與方法 29 3.1 實驗機制 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 3.2 實驗材料介紹 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 3.2.1 商用晶片材料 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 3.2.2 自製金電極製程 . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 3.2.3 生物材料介紹 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 3.2.4 CRISPR/Cas 系統相關序列及探針序列 . . . . . . . . . . . . . . . 35 3.3 實驗步驟 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 3.3.1 電極前處理 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 3.3.2 DNA 固定化 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 3.3.3 CRISPR/Cas 實驗條件 . . . . . . . . . . . . . . . . . . . . . . . . 39 3.4 電化學量測方法 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 第四章 實驗結果與討論 41 4.1 使用商用晶片用於感測 Cas12a 之剪切 . . . . . . . . . . . . . . . . . 41 4.1.1 商用晶片穩定度分析 . . . . . . . . . . . . . . . . . . . . . . . . 41 4.1.2 表面改質結果 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 4.2 Cas12a 剪切測試 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 4.2.1 CRISPR/Cas 目標 DNA 之陰性對照測試 . . . . . . . . . . . . . .45 4.2.2 Cas12a 結合緩衝液測試 . . . . . . . . . . . . . . . . . . . . . . . 47 4.2.3 實驗條件對晶片之影響 . . . . . . . . . . . . . . . . . . . . . . . 51 4.3使用自製電極選擇可用於 Cas12a 剪切之基線 . . . . . . . . . . . . .52 4.3.1 自製電極前處理方式選擇 . . . . . . . . . . . . . . . . . . . . . . 52 4.3.2 重組白蛋白與牛血清蛋白對自製電極影響比較 . . . . . . . . . . 54 4.3.3 牛血清蛋白空位阻擋條件選擇 . . . . . . . . . . . . . . . . . . . 55 4.4Hairpin DNA 探針穩定性分析與未來設計方向 . . . . . . . . . . . . 58 第五章 結論 61 參考文獻 63 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | DNA 生物 感測器 | zh_TW |
| dc.subject | CRISPR/Cas 系統 | zh_TW |
| dc.subject | 電化學 | zh_TW |
| dc.subject | 電化學阻抗圖譜 | zh_TW |
| dc.subject | 循環伏安法 | zh_TW |
| dc.subject | DNA biosensor | en |
| dc.subject | electrochemical impedance spectroscopy | en |
| dc.subject | electrochemistry | en |
| dc.subject | CRISPR/Cas system | en |
| dc.subject | cyclic voltammetry | en |
| dc.title | 應用於 CRISPR/Cas12a 之電化學阻抗量測方法研究 | zh_TW |
| dc.title | Investigation of Electrochemical Impedance-Based Detection Methods for CRISPR/Cas12a | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 吳靖宙;余靈珊;張子璿 | zh_TW |
| dc.contributor.oralexamcommittee | Ching-Chou Wu;Ling-Shan Yu;Tzu-Hsuan Chang | en |
| dc.subject.keyword | CRISPR/Cas 系統,電化學,電化學阻抗圖譜,循環伏安法,DNA 生物 感測器, | zh_TW |
| dc.subject.keyword | CRISPR/Cas system,electrochemistry,electrochemical impedance spectroscopy,cyclic voltammetry,DNA biosensor, | en |
| dc.relation.page | 72 | - |
| dc.identifier.doi | 10.6342/NTU202503313 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2025-08-11 | - |
| dc.contributor.author-college | 電機資訊學院 | - |
| dc.contributor.author-dept | 生醫電子與資訊學研究所 | - |
| dc.date.embargo-lift | 2030-08-01 | - |
| 顯示於系所單位： | 生醫電子與資訊學研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-113-2.pdf 此日期後於網路公開 2030-08-01 | 11.31 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。