臺灣博碩士論文加值系統

本研究是應用定域化表面電漿共振結合光纖的生物感測器，用以檢測與子宮頸癌高度相關的四型人類乳突病毒(Human Papillomavirus，HPV)。實驗中所使用的DNA探針型式為長50 mer，3’端利用雜合原理合成為雙股，最上方接近5’端處設計為20 mer具有特異性的HPV DNA序列。由雜交訊號結果發現，固定HPV-11 DNA探針於光纖上的最佳含浸條件為：濃度200 nM、20 mM MgCl2 + 20 mM KCl溶液、含浸10小時。當42 ℃下，此修飾好HPV-11 DNA探針與HPV-11 PCR雜交，其相對訊號可下降3.8 %，偵測極限達0.0056 ng/ml。而其它HPV-6、HPV-16與HPV-18 之DNA探針與PCR雜交反應，其相對訊號分別下降4.78 %、4.81 %與5.13 %，偵測極限分別為0.0081 ng/ml、0.004 ng/ml與0.00165 ng/ml。在四型HPV探針之專一性方面，除了HPV-11探針對HPV-18之PCR的專一性較差，其餘均有不錯的專一性。在動力學參數估計上，計算出ka約為0.9278*106 M-1S-1，kd約為0.0001206 S-1，KA約為7.693*109 M-1，代表DNA探針與PCR產物之親合力佳。故本實驗使用雙股DNA探針可以成左瑰侅PV四型之相關核酸序列，且有不錯的反應及專一性。

A reflection-based localized surface plasmon resonance fiber optic sensor has been developed to detect the 6, 11, 16, and 18 genotypes of Human papillomavirus (HPV) associated with cervical cancer. The detecting probe combines with single strand DNA 50 mer and thiolated 20 mer and annealing up to 95 oC, leading to the double-stranded formation in the terminal and designing 5’ end 20 mer which has specificity DNA sequence. The optimal immobilizing conditions of HPV-11 on to the modified optical fiber are 200nM DNA, 20 mM MgCl2 + 20 mM KCl solution, and immersing 10 h. As hybridizing with HPV-11 PCR product at 42 oC, the relative intensity change of HPV-11 DNA probe-functionalized sensor was 3.8 % with a 0.0056 ng/ml limit of detection. The responses of HPV-6、-16 and -18 DNA probes to particular PCR products are 4.78 %、4.81 % and 5.13 %, and have the 0.0081 ng/ml、0.004 ng/ml and 0.00165 ng/ml limit of detection, respectively. The HPV-11 probe has the good specificity except the HPV-18 PCR product and other three types of HPV probes have good specificity. The kinetic parameters of HPV-6, ka, kd, and KA, are 9.278x105 M-1s-1, 1.206x10-4 s-1, and 7.693x109 M-1, respectively. The result shows that the four types of HPV double strand DNA probes have a good affinity and response with PCR product.

致謝
中文摘要
英文摘要
目錄
圖目錄
表目錄
符號說明
第一章 緒論
1.1概述
1.2各章節簡介
第二章 文獻回顧
2.1 子宮頸癌和Human Papillomavirus (HPV)之相關性
2.2 Human Papillomavirus簡介
2.3 各種檢測方法
2.3.1 傳統子宮抹片檢查(巴式塗片)
2.3.2 Southern blot hybridization ( DNA ) 南方式墨點法
2.3.3 Hybrid capture, Digene HC2 test
2.3.4 PCR，核酸放大技術
2.3.5 實驗室方法檢測HPV
2.4 生物感測器
2.4.1 以DNA片段做為生物辨識元
2.5 表面電漿共振(Surface Plasmon Resonance,SPR)
2.5.1 定域化表面電漿共振原理(Localized surface plasmon resonance)
2.5.2 表面增強拉曼散射
2.5.3 光纖式表面電漿共振感測器
2.6 實驗目的
第三章 實驗材料、設備與方法
3.1 實驗藥品及儀器設備
3.1.1 實驗藥品
3.1.2 實驗儀器
3.2 實驗流程與備製方法
3.2.1 HPV PCR產物檢測 (HPV-6、11、 16、 18)
3.2.1-1 HPV-11 PCR產物檢測之實驗流程
3.2.2 雙股DNA合成示意圖
3.2.3 穿透式光纖生物感測器裝置圖
3.2.4 DNA HPV檢測光纖之製備與HPV PCR (HPV-11、6、16、18 )產物之檢測
3.2.4-1 製備HPV的雙股DNA探針
3.2.4-2 金奈米HPV檢測光纖之備製
3.2.5 製備AFM表面分析樣品
3.2.6 金奈米光纖以TE buffer固定探針濃度之討論
3.2.7 金奈米光纖以MgCl2 solution固定探針濃度之討論
3.2.8 金奈米光纖以MgCl2 sloution固定探針時間之討論
3.2.9 金奈米光纖以不同離子濃度為固定液之固定DNA探針討論
3.2.10 HPV-11 DNA光纖以不同的雜交液雜合之效率討論
3.2.11 HPV-6、16、18型DNA金奈米光纖之較佳化條件之討論
3.3 金奈米光纖修飾四型HPV DNA探針之專一性討論
3.3.1 金奈米光纖修飾四型HPV DNA探針之單獨專一性之討論
3.4 DNA動力學推導
3.4.1實驗原理推

第四章 結果與討論
4.1 HPV-11 PCR產物之檢測
4.1.1 20 mer單股DNA(ssDNA)探針檢測HPV-11 PCR產物(約3000 base)之討論
4.1.2 20 mer單股DNA + 6 mer T鹼基探針檢測HPV PCR 產物 (約200 base)之討論
4.1.3 雙股DNA探針檢測HPV PCR產物(約200 base)之討論
4.2利用AFM分析表面形貌之改變
4.3 HPV-11型PCR產物之檢測
4.3.1 金奈米光纖以TE buffer浛浸雙股HPV-11 DNA探針之濃度討論
4.3.2 金奈米光纖以MgC12 + KCl sloution含浸雙股HPV-11 DNA探針之濃度討論
4.3.3金奈米光纖含浸HPV-11雙股DNA探針之固定化溶液中改變 Mg2+與KCl含量之討論
4.3.4 金奈米光纖含浸HPV-11雙股DNA探針之固定化時間討論
4.3.5 HPV-11型DNA光纖檢測PCR產物時改變雜交buffer之討論
4.3.6 HPV-11型DNA光纖檢測PCR產物時加入Tween 20之討論
4.3.7 備製HPV-11雙股DNA光纖較佳條件下檢測PCR產物
4.4 HPV-6型PCR產物之檢測
4.4.1 HPV-6 型DNA探針較佳修飾條件之探討
4.4.2改變固定DNA探針溶液中KCl含量於HPV-6型DNA探針
4.5 HPV-16型與HPV-18 PCR產物之檢測
4.6 比較四型HPV-6、11、16、18型較佳固定濃度之相關性
4.7 四型HPV DNA探針相互之專一性討論
4.8 探討DNA雜交動力學
4.8.1 DNA雜交動力學參數估計
第五章 總結
5.1 HPV DNA 探針之生化感測器
5.2 未來展望

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