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研究生:林文智
研究生(外文):Wen-Zhi Lin
論文名稱:發展以表面功能化之金奈米粒子為基礎的比色法應用於過氧化氫和生物素檢測
論文名稱(外文):Using Biofunctionalized Gold Nanoparticles to Develop a Colorimetric Biosensing System for the Detection of Hydrogen Peroxide and Biotin
指導教授:侯劭毅侯劭毅引用關係
指導教授(外文):Shao-Yi Hou
口試委員:劉正哲王勝仕蔡文銓黃光策陳正忠黃志宏
口試日期:2018-07-09
學位類別:博士
校院名稱:國立臺北科技大學
系所名稱:工程學院工程科技博士班
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:中文
論文頁數:62
中文關鍵詞:比色法免疫微米磁珠生物素過氧化氫金奈米粒子DNA
外文關鍵詞:colorimetric detectionimmunomagnetic microbeadsbiotinhydrogen peroxidegold nanoparticlesDNA
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金奈米粒子因其特殊的光學、化學、生物相容性等特質,且可和抗體以物理作用力吸附或與經過硫醇修飾的核酸片段形成硫金鍵結作為探針,而應用在生物醫學方面的相關檢測。在人體的許多生理反應均有活性氧化物質的參與,然而當細胞生長環境中含有過量的上述物質時,反而對人體有害;另外,半抗原是一種小分子,它包括生物素、一些濫用藥物及存在於一些食物中的毒素。然而,針對上述物質的檢測方法大多耗時且不便,因此,本研究將以表面功能化之金奈米粒子為基礎的比色法應用於上述物質的檢測。
本研究可分為兩大部分,第一部分為利用過氧化氫當目標分子,以DNA修飾的金奈米粒子與芬頓反應(Fenton reaction)之比色法方式來實施檢測。結果顯示偵測極限值為1 μM且檢測時間為15分鐘。第二部分為利用生物素當目標分子,以生物素修飾的金奈米粒子與抗生物素抗體修飾的免疫微米磁珠,藉磁性分離以競爭式的比色法來實施檢測。結果顯示偵測極限值為2 pmol且檢測時間為15分鐘。
本研究可用肉眼直接判讀結果,不需要特殊儀器來實施檢測。所以本實驗具有快速、明確及操作方便等優點,將有助於第一線人員實施快篩檢測。
Gold nanoparticles (AuNPs) have been extensively used in different applications over the past decades, because of their easily controllable size distribution, long-term stability, ease of synthesis and surface functionalization by proteins and nucleotides. Reactive oxygen species (ROS) is involved in many biological reactions. However, excessive ROS is detrimental for human health. Haptens are small molecules with low molecular weight that include biotin, abused drug and many toxins in food. In addition, various methods have been developed for ROS and hapten. However, complex procedures, time-consumption, high equipment cost and personnel training, limit their use in resource-limited laboratories. Based on the above, we used biofunctionalized gold nanoparticles for the detection of ROS and hapten.
In the first part of our study, we used H2O2 as a model molecule and DNA-modified AuNPs as a probe for the colorimetric detection of ROS. The results showed that the limit of detection (LOD) was 1 μM and the detection time was 15 minutes. In the second part of our study, we used biotin as a model molecule and anti-biotin antibody-modified magnetic beads (Ab-MBs) and biotinylated thiol-DNA gold nanoparticles (biotin-GNPs) for competitive colorimetric detection of hapten. The detection limit was found to be 2 pmol and the assay could be completed quickly, taking only 15 minutes. Based on these results, the proposed assay needs no tedious pretreatment and the results can be observed by naked eyes. Therefore, this simple and rapid assay has potential application for the first-line to detect various analytes.
摘 要 I
ABSTRACT II
誌 謝 III
目 錄 IV
表目錄 VI
圖目錄 VII
第一章 緒論 1
1.1金奈米粒子(Gold nanoparticles, AuNPs)的簡介 1
1.1.1金奈米粒子的合成 1
1.1.2金奈米粒子的光學特性 2
1.1.3金奈米粒子的表面功能化 3
1.1.4金奈米粒子的比色法原理 3
1.1.5金奈米粒子與DNA的結合應用 5
1.1.6金奈米粒子比色法的應用 7
1.2活性氧化物質與過氧化氫簡介 12
1.3半抗原(Hapten)與生物素(Biotin)簡介 14
1.4檢測過氧化氫及生物素的方法 15
1.5研究動機及目的 15
第二章 實驗方法與實驗流程 16
2.1實驗架構 16
2.1.1過氧化氫檢測 16
2.1.2生物素檢測 17
2.2實驗材料與試劑 18
2.2.1過氧化氫檢測之實驗材料與試劑 18
2.2.2生物素檢測之實驗材料與試劑 19
2.3實驗儀器與設備 20
2.3.1過氧化氫檢測之實驗儀器與設備 20
2.3.2生物素檢測之實驗儀器與設備 22
2.4過氧化氫檢測之DNA探針設計 22
2.5雙硫單股寡核苷酸之還原步驟 23
2.6將thiol-DNA修飾到金奈米粒子表面的實驗步驟 23
2.7過氧化氫檢測的實驗步驟 24
2.8生物素檢測的實驗步驟 25
第三章 結果與討論 26
3.1過氧化氫的檢測 26
3.1.1奈米金的表面功能化測試 26
3.1.2芬頓反應對DNA修飾的金奈米粒子之影響 27
3.1.3實驗條件最佳化 28
3.1.4在最佳化條件下對過氧化氫實施比色法檢測 34
3.2生物素檢測 38
3.2.1實驗條件最佳化 38
3.2.2在最佳化條件下對生物素實施比色法檢測 41
3.2.3應用於食品樣本之檢測 44
第四章 結論 46
參考文獻 47
附 錄 52
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