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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76436完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 何佳安 | |
| dc.contributor.author | Lik-Bin Chong | en |
| dc.contributor.author | 張立斌 | zh_TW |
| dc.date.accessioned | 2021-07-09T15:52:19Z | - |
| dc.date.available | 2022-09-12 | |
| dc.date.copyright | 2017-09-12 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-08-17 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76436 | - |
| dc.description.abstract | 雖然胰臟癌的發生事件比起其它癌症來得低，可是它卻是造成癌症相關死亡事件的第四名殺手。由於罹患胰臟癌的病人不會表現出特異的性狀，且胰臟癌的的偵測與診斷存在一定的困難性，因此胰臟癌確診病人的五年存活率只有6%左右。根據先前的文獻指出，罹患上胰臟癌的病人會大量表現某些特定循環性的微型核醣核酸 (microRNAs)，並可在病人血液中被偵測出。在本研究中，我們應用酵素驅動型核酸分子奈米機器為放大策略，發展用於檢測胰臟癌相關之生物標記分子miR-221的電化學生化感測平臺。此平臺主要含蓋三部分，分別為⑴. 聚合酶輔助目標分子之循環反應 (Polymerase-assisted Target Recycling)、⑵. 外切酶剪切反應 (Exonuclease Digestion) 和⑶. 電化學訊號的輸出 (Electrochemical Signal Output)。我們首先以膠體電泳的方式先進行產物初步之分析，證明當受質Hairpin (H)、目標分子miR-221 (M)、聚合酶 (P)、外切酶 (E) 四大元件缺乏其一時，DNA Nanomachine則無法運作，最終產物P2就無法成功產出。接著我們進行一系列條件之最佳化，其中包括緩衝溶液的選擇、受質使用的濃度、聚合酶和剪切酶的濃度比，以獲得最多的最終產物P2。在電化學偵測的部分，我們先確認最終產物P2可以成為誘導股，並與修飾在工作電極表面亞甲藍標定的探針進行雜合，進而造成電化學訊號之改變。接著我們進行阻隔劑 (Blocking agent) 和鍍金時間之最佳化，以得到最佳之實驗操作條件。藉由標準曲線 (Calibration curve) 的結果顯示，我們所設計的電化學生化感測器的偵測極限為9.6 pM。我們將20 fmol的miR-221添加入血清中，所測得之回收率 (Recovery rate) 接近百分之百。該感測平台被證實具有可偵測血清中之microRNA之可行性和潛力。此外，我們也使用不同分析物 (目標分子和其同源家族成員) 進行DNA Nanomachine之選擇性測試，結果顯示所研發的電化學感測器只會針對目標分子miR-221展現出訊號之改變，從而證實我們所研發的電化學感測器具有良好之選擇性。 | zh_TW |
| dc.description.abstract | Pancreatic cancer with a single-digit survival rate, at ~7%, has the highest mortality rate among all cancers. Similar to lung cancer, pancreatic cancer patients seldom exhibit disease-specific symptoms until late disease stage; therefore an establishment of early detection method for pancreatic cancer is important to classify pancreatic cancer at earlier stages. MicroRNAs (miRNAs) are a class of highly conserved non-coding RNA consisted of approximately 22 nucleotides that regulate gene expression by either degradation of mRNAs or translational repression at the post-transcriptional level. MicroRNAs are voted as potential biomarkers for therapeutics and point-of-care diagnostics, as their aberrant expressions are always associated with diseases and even various cancers. In this study, a novel isothermal electrochemical biosensor was designed for sensitive and selective detection of circulating pancreatic cancer-related microRNA. The amplification strategy was based on dual enzyme-empowered target recycling performed by polymerase and exonuclease. Initially, the presence of target miRNA selectively unfolded a hairpin probe (H), followed by the polymerase-triggered elongation of the H along with the strand displacement of target miR. While the target miR went on a new cycle of H unfolding, a blunt end of the He was formed and digested by the exonuclease, resulting in the release of intermediate DNA product (P1). The released P1 was able to hybridize with a new H, initiating a new round of exonuclease-assisted digestion of H, yielding a final DNA product (P2). Finally, a “signal-off” electrochemical biosensor was established when the signal inducer P2 hybridized with a methylene blue-labelled complementary hairpin probes (P2C) that immobilized on a gold nanostructured screen printed electrode (AuNC@SPCE) through gold-thiol bond. Under optimal condition, this biosensor offered an excellent sensitive platform towards the detection of target miR, with detection limit of 25 pM together with a dynamic range of six orders of magnitude. In addition, an excellent selectivity was achieved in discriminating its homologous inter-family members. We have herein successfully demonstrated a biosensing platform for the detection of pancreatic cancer-related miR and the biosensor holds a potential in the early diagnosis of pancreatic cancer to improve the survival rate in the future. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-09T15:52:19Z (GMT). No. of bitstreams: 1 ntu-106-R04b22045-1.pdf: 8326487 bytes, checksum: d1a54c80b36ebd7cd11a5485e0b2b206 (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 第一章 緒論 1
1.1. 胰臟癌 (Pancreatic cancer) 1 1.1.1. 認識胰臟 1 1.1.2. 人類胰臟癌 1 1.1.3. 胰臟癌之診斷 4 1.2. 微型核醣核酸 (MicroRNA) 5 1.2.1. miRNA生合成與作用機制 5 1.2.2. miRNA與癌症 8 1.2.3. 循環性miRNA (Circulating microRNA) 10 1.2.4. miR-221做為胰臟癌之生物指標 11 1.2.5. miRNA之偵測方法 13 1.3. 脫氧核醣核酸奈米機器 (DNA Nanomachine) 19 1.3.1. 以鏈交換反應為基礎之DNA奈米機器 19 1.3.2. 溫度調控之DNA奈米機器 20 1.3.3. 酵素驅動型奈米機器 21 1.4. 等溫核酸放大技術 (Isothermal Amplification) 22 1.4.1. 酵素驅動型等溫核酸放大技術 22 1.4.2. 非酵素驅動型核酸放大技術 25 1.5. 酵素輔助核酸目標循環 (Enzyme-assisted Target Recycling) 27 1.5.1. 典型之輔助目標物循環法 30 1.5.2. 5’→3’外切酶分析法 30 1.5.3. 入侵分析法 (Invader Assay) 31 1.5.4. Lambda exonuclease 輔助目標分子循環法 32 1.6. DNA生化感測器 (DNA Biosensor) 33 1.6.1. 生物元件之修飾法 33 1.6.2. 電化學生化感測器 35 1.7. 電化學偵測法 37 第二章 實驗材料與方法 39 2.1. 核酸序列 39 2.2. 實驗試劑與材料 41 2.3. 實驗儀器 44 2.4. 緩衝溶液 46 2.5. 聚丙烯醯胺膠體電泳 (Polyacrylamide gel electrophoresis, PAGE) 47 2.6. 酵素驅動型核酸奈米機器 (DNA Nanomachine) 產物之鑑定 48 2.7. Hairpin 5’端overhang鹼基数目之最佳化 48 2.8. 反應緩衝溶液之最佳化 49 2.9. 受質H濃度之最佳化 49 2.10. P2C (Complementary Sequence of P2) 之最佳化 50 2.11. 酵素濃度比例之最佳化 50 2.12. 選擇性 (Selectivity) 之測試 51 2.12.1. 受質H之專一性測試 51 2.12.2. KF之專一性延長測試 51 2.12.3. λ之專一性剪切測試 51 2.12.4. P2C之專一性測試 51 2.13. 鍍金時間 (Gold electrodeposition time) 之最佳化 53 2.13.1. 以Fe(CN)63-/4-為電化學訊號分子 53 2.13.2. 以P2C-MB探針上之MB為電化學訊號之分子 56 2.14. P2C-MB固定化濃度之最佳化 59 2.15. P2C-MB於工作電極之密度鑑定 62 2.16. 阻隔劑 (Blocking agent) 之最佳化 65 2.16.1. 以經不同阻隔劑處理之電極量測不同濃度亞甲藍溶液之校正曲線所得之slope和R2進行比較 65 2.16.2. 經不同阻隔劑處理之電極對BSA非專一性吸附之抗性 67 2.17. 工作電極表面之鑑定 70 2.18. 電化學生化感測器應用於目標分子miR-221之分析 71 2.19. 電化學生化感測器之選擇性測試 72 2.20. 儲存天數 (Storage time) 對於氧化電化學訊號之影響 73 2.21. 同日與隔日目標分子miR-221濃度準確度和精密度之比較 76 2.22. 血清 (Serum) 中目標分子miR-221之回收率 (Recovery rate) 77 第三章 實驗結果與討論 79 3.1. 實驗設計 79 3.2. 酵素驅動型核酸奈米機器 (DNA Nanomachine) 產物之鑑定 86 3.3. Hairpin 5’端overhang鹼基数目之最佳化 90 3.4. 反應緩衝溶液之最佳化 95 3.5. 受質H濃度之最佳化 99 3.6. P2C (Complementary Sequence of P2) 之最佳化 101 3.7. 酵素濃度比例之最佳化 105 3.8. 選擇性 (Selectivity) 之測試 109 3.8.1. 受質H之專一性測試 110 3.8.2. KF之專一性延長測試 112 3.8.3.. λ之專一性剪切測試 114 3.8.4. P2C之專一性測試 116 3.9. 網版印刷碳電極工作電極製備條件之最佳化 118 3.9.1. 鍍金時間 (Gold electrodeposition time) 之最佳化 118 3.9.2. P2C-MB固定化濃度之最佳化 123 3.9.3. P2C-MB於工作電極之密度鑑定 123 3.9.4. 阻隔劑 (Blocking agent) 之最佳化 128 3.9.5. 工作電極表面之鑑定 135 3.10. 電化學生化感測器應用於目標分子miR-221之分析 137 3.11. 電化學生化感測器之選擇性測試 142 3.12. 儲存天數 (Storage time) 對於氧化電化學訊號之影響 144 3.13. 同日與隔日目標分子miR-221濃度準確度和精密度之比較 148 3.14. 血清 (Serum) 中目標分子miR-221之回收率 (Recovery rate) 151 第四章 結論 153 第五章 參考文獻 155 | |
| dc.language.iso | zh-TW | |
| dc.title | 以酵素驅動型核酸分子奈米機器為放大策略發展快速篩檢胰臟癌相關的微小核醣核酸片段之電化學生化感測器 | zh_TW |
| dc.title | Electrochemical Biosensor for Pancreatic Cancer-specific MicroRNA Based on Enzyme-powered DNA Nanomachine Amplification Strategy | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 徐士蘭,楊家銘,鄭建中,廖明淵,陳平 | |
| dc.subject.keyword | 胰臟癌,微型核醣核酸,電化學生化感測器,聚合?,外切?, | zh_TW |
| dc.subject.keyword | pancreatic cancer,microRNA,methylene blue,screen printed electrode, | en |
| dc.relation.page | 168 | |
| dc.identifier.doi | 10.6342/NTU201703715 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2017-08-18 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| dc.date.embargo-lift | 2022-09-12 | - |
| 顯示於系所單位： | 生化科技學系 | |
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