近年來因抗生素的濫用，導致細菌產生了抗藥性的變異，已成
為公共衛生安全的一大危險。本實驗以磁性奈米粒子結合具有高特異
性的核酸適體（Aptamer）捕捉具有抗藥性的細菌，耐甲氧西林金黃
色葡萄球菌（methicillin-resistant S.aureus,MRSA）。本研究之材料具
有易合成，和良好收集之特性，於表面修飾二氧化矽（SiO2）和聚丙
烯酸(PAA)，改變磁性奈米粒子上的官能基，修飾上對 MRSA 具有
高特異性的單股 DNA。此檢測方法主要為使用紫外光光譜儀及螢光
儀檢測液體樣品加入磁性奈米粒子前後的吸收值，可以快速地由液體
樣品中篩選出抗藥性細菌，成本也比以往抗體偵測方法低。此材料也
具有高的特異性，相同的細菌濃度之下，用於抗藥性的金黃色葡萄球
菌 60 分鐘的捕捉時間，即有 95%以上的捕捉率；用於非抗藥性的金
黃色葡萄球菌，60 分鐘的捕捉率，卻只有不到 15%的捕捉率；以 0.1M
NaCl / 1% NH4OH 作為洗脫液可將 MNP@Aptamer 捕捉的 MRSA 洗
脫且結合質譜儀鑑定細菌種類，MNP@Aptamer 也可重複使用達數十
次。

In recent years, the abuse of antibiotics has led to bacterial variation in drug resistance, which has become a major risk for public health safety.
The present work applied magnetic nanoparticles modified with highly specific aptamers to the capture of antibiotic-resistant bacteria, methicillin resistant Staphylococcus aureus (MRSA). The affinity probe is easy to synthesize and reusable. After silica and polyacrylic acid was modified on the surface of magnetic nanoparticles, and the highly specific DNA of MRSA was covalently bound to the particles. Antibiotic-resistant bacteria can be quickly captured by the probes. The probe is superior to antibody probes in stability and cost. The 60-minute capture time for MRSA has a capture rate of more than 90% while the capture rate for the antibiotic-susceptible Staphylococcus aureus is less than 15%.

目錄
壹、緒論 1
1、前言 1
2、細菌之抗藥性 2
3、傳統鑑定微生物方法 3
3.1、直接鏡檢計數法（Direct count） 4
3.2、菌落計數法（Colony counting） 5
3.3、濾膜計數法（membrance filter method） 6
3.4、混濁度測試法（tutbidity measurement） 7
4、抗藥性細菌檢測 8
4.1、傳統抗生素敏感性測試（antimicrobial susceptibility test） 8
4.2、聚合酶連鎖反應測定 10
4.3、質譜檢測法 11
4.4、金奈米粒子測定法 12
4.5、核酸適體與氧化石墨烯感測器測定法 12
4.6、上轉換奈米粒子與DNA適體檢測病原菌 13
5、細菌介紹 15
5.1、金黃色葡萄球菌 15
5.2、耐甲氧西林金黃色葡萄球菌 16
6、磁性奈米粒子 19
6.1、磁性奈米粒子之製備 19
6.2、磁性奈米粒子的發展及應用 19
7、適體（Aptamer） 22
7.1、適體（Aptamer）篩選 22
7.2、適體（Aptamer）應用及發展 25
8、質譜儀與細菌之研究 29
9、研究動機 32
貳、研究內容 33
1、儀器與藥品 33
1.1、 儀器 33
1.2、 藥品 33
1.3、儀器參數與條件 34
1.4、資料庫搜尋參數設定 36
2、實驗方法 36
3、實驗步驟 37
3.1、氧化鐵(Fe3O4)奈米粒子合成 37
3.2、合成表面修飾二氧化矽（SiO2）之磁性奈米粒子 37
3.3、合成表面修飾聚丙烯酸（PAA）之磁性奈米粒子 37
3.4、奈米粒子定量 38
3.5、合成表面修飾適體（aptamer）之磁性奈米粒子 38
3.6、細菌樣品製備 40
3.7、核酸適體修飾鐵奈米粒子檢測細菌樣品 40
3.8、核酸適體修飾鐵奈米粒子洗脫細菌樣品 41
3.9、細菌蛋白質萃取與消化 41
參、結果與討論 45
1、磁性奈米粒子修飾 (MNP@SiO2@PAA) 之官能基鑑定 45
2、聚丙烯酸修飾鐵奈米粒子與核酸適體結合優化 46
2.1、聚丙烯酸修飾鐵奈米粒子與核酸適體結合時間優化 46
2.2、聚丙烯修飾鐵奈米粒子與核酸適體結合比例優化 47
3、以FITC染色MRSA 48
4、核酸適體修飾鐵奈米粒子捕捉MRSA優化 49
4.1、MNP@Aptamer捕捉MRSA適體優化、捕捉溫度、時間 49
4.2、MNP@Aptamer 比例優化捕捉MRSA 52
4.3、MNP@Aptamer 捕捉不同濃度MRSA 54
5、MNP@Aptamer 特異性測試 55
5.1、MNP@Aptamer分別捕捉S.aureus、E.coli時間及捕捉率 55
5.2、MNP@Aptamer捕捉分別S.aureus及E.coli不同細菌量測試特異性 56
6、MRSA洗脫於核酸適體鐵奈米粒子 57
6.1、洗脫液優化 57
6.2、洗脫不同濃度MRSA 58
6.3、捕捉MRSA後不同重量MNP@Aptamer洗脫率 59
7、混和菌種測試特異性捕捉 60
8、磁性奈米粒子結合核酸適體使用次數 62
9、LC-MS/MS分析細菌的蛋白質水解產物 64
肆、結論 67
伍、參考文獻 68
陸、附錄 74

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