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論文中文名稱:以金奈米粒子檢測活性氧化物質之呈色法 [以論文名稱查詢館藏系統]
論文英文名稱:Colorimetric detection of reactive oxygen species using gold nanoparticles [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生化與生醫工程研究所
畢業學年度:105
畢業學期:第一學期
中文姓名:梁智開
英文姓名:LIANG CHIH-KAI
研究生學號:103688012
學位類別:碩士
口試日期:2017/01/16
論文頁數:45
指導教授中文名:侯劭毅
口試委員中文名:黃光策;王勝仕;許哲奇;劉正哲;侯劭毅
中文關鍵詞:活性氧化物質DNA金奈米粒子芬頓反應法過氧化氫氫氧自由基呈色法
英文關鍵詞:reactive oxygen speciesDNAgold nanoparticlesfenton reactionhydrogen peroxidehydroxyl radicalcolorimetric detection
論文中文摘要:許多生理代謝反應均有活性氧化物質(reactive oxygen species, ROS)的參與,因此對活體細胞來說,ROS是很重要的物質。然而當細胞生長環境中含有過量的ROS時,ROS反而對細胞有危害性。本實驗中,利用修飾過DNA的金奈米粒子與芬頓反應法(Fenton reaction),以呈色法方式檢測過氧化氫(H2O2)。實驗分成兩步驟,第一步驟是透過芬頓反應法,讓鐵二價離子與H2O2反應生成氫氧自由基。第二步驟則加入修飾過DNA的奈米金,利用氫氧自由基過多會攻擊DNA,使其斷裂的特性。減少附著在奈米金上DNA的鹼基數量,使得奈米金表面的負電荷減少,進而產生奈米金的聚集現象,溶液的顏色從紅轉變成紫。此方法的檢測極限為1µM且反應時間只需要15分鐘,可用肉眼直接判斷結果,不需要特殊儀器偵測。此研究具有快速、操作方便、低成本等優點,是一種有潛力的檢測法。
論文英文摘要:Reactive oxygen species (ROS) are involved in many metabolic pathways. Hence, ROS is important for living cells. However, ROS which exceed the limit will be hazardous. In this study, we used DNA modified gold nanoparticles and Fenton reaction to develop a two-steps colorimetric detection for hydrogen peroxide (H2O2). In step 1, we used ferrous ion and hydrogen peroxide to produce hydroxyl radical (OH·) by Fenton reaction. In step 2, DNA modified gold nanoparticles were added and hydroxyl radical attacked DNA. It breaks phosphodiester bond on DNA and decreases the quantity of DNA on the surface of gold nanoparticles. This decrease caused gold nanoparticles aggregation. The color of solution changed from red to purple. In this study, the detection limit is 1μM and it takes 15 minutes for detection. We can directly determine the results by naked eye and without any instrument. This method is fast, easy operation and low-cost. To be a potential assay.
論文目次:目錄
摘 要 I
ABSTRACT II
誌 謝 III
目錄 IV
表目錄 VII
圖目錄 VIII
第一章 緒論 1
1.1活性氧化物質與過氧化氫介紹 1
1.2現行檢測活性氧化物質方法 4
1.2.1呈色檢測法 4
1.2.2電化學檢測法 5
1.2.3螢光檢測法 5
1.4芬頓反應(Fenton reaction) 6
1.5金奈米粒子介紹 7
1.5.1膠體金 7
1.5.2金奈米粒子結構 7
1.5.3金奈米粒子特性 8
1.5.3.1表面積激增 8
1.5.3.2電解質敏感性 8
1.5.3.3硫金鍵結 9
1.5.3.4光學特性 9
1.5.4金奈米粒子與DNA的結合與應用 11
1.6 研究策略 13
第二章 實驗方法與流程 14
2.1實驗架構 14
2.2實驗儀器與設備 15
2.3實驗藥品 17
2.4單股寡核苷酸之設計 18
2.5修飾雙硫單股寡核苷酸到金奈米粒子表面 19
2.5.1雙硫單股寡核苷酸還原與純化 19
2.5.2硫醇單股寡核苷酸修飾到金奈米粒子表面 20
2.6檢測過氧化氫之實驗步驟 21
第三章 結果與討論 22
3.1金奈米粒子-硫醇單股寡核苷酸之性質測試 23
3.1.1吸收光譜 23
3.1.2電解質耐受性 24
3.2芬頓反應對金奈米粒子-硫醇單股寡核苷酸的電解質耐受性之影響 26
3.3 TE buffer對芬頓反應的影響 28
3.4條件的最佳化 31
3.4.1 Fe2+濃度的最佳化 31
3.4.2步驟一反應時間的最佳化 33
3.4.3步驟二反應時間的最佳化 35
3.5檢測過氧化氫 37
第四章 結論 42
參考文獻 43
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