近年來食安問題越來越受到民眾以及政府機關的重視，因此能快速分析食品中致病菌成為我們重視的一環。本實驗合成三種微米級孔洞材料，第一種為修飾聚乙烯亞胺 (polyethylenimine, PEI) 的多孔性材料 (poly (GMA-EDMA@PEI))，經水浴加熱縮合後形成；第二種為修飾氧化鋯多孔矽材料，經高溫鍛燒製成；第三種則為氧化鋯微米級孔洞性材料，主體結構為氧化鋯，雖然結構沒有多孔矽材料堅固，但可以承受的pH值範圍較多孔矽材料大，以上材料利用不同蛋白質在等電點的差異，利用改變溶液pH值達到分離蛋白質的效果，優化且延伸對細菌的捕捉，以pH 7.6 Tris-HCl buffer當結合、清洗液，pH 11 ACN/NH4OH混合液作為洗脫液，以細胞計數的方式對大腸桿菌進行捕捉，poly (GMA-EDMA@PEI) 最大捕捉量為7.1×1010個細菌/mL，最低捕捉極限則為6.6個細菌/mL；ZrO2/ SiO2最大捕捉量為8.5×109個細菌/mL，最低捕捉極限則為1.3×102個細菌/mL；zirconia monolithic最大捕捉量為1.0×1010個細菌/mL，最低捕捉極限則為1.4×102個細菌/mL，經管住捕捉後的細菌以酵素水解，並結合質譜儀鑑定細菌種類，因此可應用於食品中微量細菌的檢測。

目錄
壹、緒論 1
1、前言 1
2、傳統鑑定微生物方法 2
2.1、直接鏡檢計數法 (Direct count) 3
2.2、菌落計數法 (Colony counting) 4
2.3、濾膜計數法 (Membrane filter method) 5
2.4、混濁度測定法 (Turbidity measurement) 6
3、大腸桿菌 7
4、金黃色葡萄球菌 10
5、整體柱 (Monolithic) 管柱 11
5.1、多孔洞材料 11
5.2、有機多孔洞材料的發展 13
5.3、微米級多孔洞材料與蛋白質作用力 16
5.4、微米級多孔洞材料在細菌捕捉之應用 17
6、質譜儀在細菌鑑定上之應用 20
7、研究動機 23
貳、研究內容 25
1、儀器與藥品 25
1.1、儀器 25
1.2、藥品 26
1.3、儀器使用方法 27
1.4、儀器參數與條件 29
1.5、資料庫搜尋參數設定 30
2、實驗步驟 31
2.1、微米級孔洞性材料整體柱合成與修飾 31
2.2、合成微米級孔洞性材料整體柱管柱修飾氧化鋯 (zirconium oxide-silica, ZrO2/SiO2) 36
2.3、合成氧化鋯微米級孔洞性材料整體管柱 (zirconia monolith) 40
2.4、細菌蛋白質萃取與消化 44
參、結果與討論 49
1、微米級多孔洞材料整體柱管柱修飾聚乙烯亞胺 (poly (GMA-EDMA@PEI)) 49
1.1、經修飾聚乙烯亞胺的整體柱的表面電位 49
1.2、蛋白質吸附實驗 50
1.3、poly (GMA-EDMA@PEI) 管柱合成條件 52
1.4、不同合成比例管柱捕捉大腸桿菌及金黃色葡萄球菌 55
1.5、poly (GMA-EDMA@PEI) 管柱SEM圖 57
1.6、洗脫液優化 59
1.7、管柱捕捉細菌使用次數測試 63
1.8、poly (GMA-EDMA@PEI) 管柱捕捉其他種細菌之效果 64
1.9、管柱吸附不同濃度大腸桿菌的捕捉率和洗脫率 66
1.10、管柱捕捉不同濃度混合菌液 68
2、微米級孔洞性材料整體柱管柱修飾氧化鋯 (ZrO2/SiO2) 70
2.1、ZrO2/SiO2管柱優化條件 70
2.2、ZrO2/SiO2管柱吸附不同濃度大腸桿菌的捕捉率和洗脫率 72
3、氧化鋯微米級孔洞性材料整體管柱 (zirconia monolithic) 73
3.1、Zirconia monolithic管柱優化條件 73
3.2、Zirconia monolithic管柱吸附不同濃度大腸桿菌的捕捉率和洗脫率 75
4、poly (GMA-EDMA@PEI) 管柱、ZrO2/SiO2管柱、zirconia monolithic管柱最大捕捉量及捕捉極限之比較 76
5、MALDI-TOF MS分析細菌的蛋白質水解產物 78
5.1、MALDI-TOF MS分析大腸桿菌的蛋白質水解產物 78
5.2、MALDI-TOF MS分析金黃色葡萄球菌的蛋白質水解產物 83
6、LC-MS/MS分析細菌的蛋白質水解產物 88
6.1、LC-MS/MS分析大腸桿菌的蛋白質水解產物 88
6.2、LC-MS/MS分析金黃色葡萄球菌的蛋白質水解產物 92
6.3、LC-MS/MS分析混合細菌的蛋白質水解產物 95
肆、結論 97
伍、參考文獻 99
陸、附錄 109

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