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研究生:邱天安
研究生(外文):Tien-An Chiu
論文名稱:利用生物功能化奈米粒子快速偵測結核桿菌
論文名稱(外文):Rapid detection of mycobacteria using biofunctionalized nanoparticles and flow cytometry
指導教授:張書奇張書奇引用關係
指導教授(外文):Shu-Chi Chang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:環境工程學系所
學門:工程學門
學類:環境工程學類
論文種類:學術論文
畢業學年度:96
語文別:中文
論文頁數:112
中文關鍵詞:量子粒結核桿菌流式細胞儀
外文關鍵詞:quantum dotmycobacteriaflow cytometry
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自從19世紀德國柯霍博士發現了結核桿菌,人們開始重視這流行千年的疾病。經研究發現環境中的水體、食物、氣膠、落塵、泥土中皆有結核桿菌的存在。在1995年,全球共有三百一十萬人死於結核病;而在台灣地區,結核病為前十五大死因中的第十二位。
Mycobacterium immunogenum 為結核桿菌屬之一種,近來被發現是會引起過敏性肺炎的機會性致病菌,且類似菌種被發現存在於游泳池、飲用水配水系統、溫泉浴場(SPA)、金屬切削液製程等。
因此快速檢測及準確的鑑別環境中之結核桿菌(Mycobacteria)是非常重要的。但是,由於結核桿菌通常生長緩慢且容易聚集,以傳統的方式鑑定或量化時,很容易被忽略或低估其數量。若缺乏適當的前處理,準確的量測是相當困難的。在一些較複雜的系統中,如沉澱池水樣與金屬切削液中,使用光學儀器直接去偵測,可能發生嚴重之光學干擾而影響偵測結果。
本研究採用超音波震盪分散之前處理,並藉由流式細胞儀、生物功能化量子粒(Biofunctionalized quantum dot),以快速偵測結核桿菌,檢測時間約三小時比傳統耗時4 ~ 6天的培養方法縮短約96 ~ 97%。從實驗結果中,可以發現利用流式細胞儀配合生物功能化量子粒,可有效降低螢光干擾及維持較高之專一性,應用於結核桿菌的鑑別是確實可行的。此研究利用隨機性抗體結合方式,比較改良式免疫螢光法與生物功能化量子粒之標定率及專一性。本研究方法除可應用於結核桿菌檢測之外,也可應用於其他容易聚集及形成生物膜的微生物之快速偵測。
After Dr. Robert Koch found mycobacteria in 19th century, the world started to pay attention of this bacterial species. Environmental mycobacteria are common in all natural ecosystems, including water, soils, foods, dusts, and aerosols. In 1995, there were three million and five hundred thousand people died because of tuberculosis. It is also the top 12th death cause in Taiwan. Mycobacteria are the most likely causative agents for human pulmonary disease in metalworkers and have been found in drinking water distribution systems, water damaged buildings, SPAs or swimming pools, cooling tower, and metalworking fluids. From an environmental health perspective, it is imperative to accurately identify and quantify mycobacteria in environmental samples. However, they are slow growing and traditional culturing methods usually take days to weeks for identification and quantification. Therefore, they are easily underestimated or neglected. Furthermore, due to their aggregate-forming characteristics in environmental samples, without proper pretreatment, accurate and consistent identification and enumeration is impossible. If they are in complex matrices, such as metalworking fluids or sediments, direct optical detection is severely hindered. In this study, by employing probe sonication, flow cytometry and biofunctionalized quantum dots, labeling efficiency and specificity between a modified immunofluorescence method and a biofunctionalized quantum dot method were compared. By using biofunctionalized quantum dot, the turn-around time has been shortened more than 95%. These results strongly suggested that coupling flow cytometry with biofunctionalized quantum dots is a feasible method on rapid detection, identification, and quantification of mycobacteria and other aggregate forming bacteria in complex matrices.
摘要 i
Abstract ii
目錄 iii
圖目錄 vii
表目錄 xi
語彙 xii
第一章 前言 1
1-1 研究背景 1
1-2 研究目地 3
第二章 文獻回顧 6
2-1 環境中的結核桿菌 6
2-1-1 水體中的結核桿菌 7
2-1-2 工業製程中的結核桿菌 8
2-2 Mycobacterium immunogenum 簡介 9
2-2-1 Hypersensitivity Pneumonitis與金屬切削液 10
2-2-2 Mycobacterium immunogenum 檢測 12
2-3 Pseudomonas aeruginosa 簡介 14
2-4 流式細胞儀(Flow Cytometer) 15
2-4-1 流式細胞儀演進 16
2-4-2 基本原理 17
2-5 量子粒 22
2-5-1 量子粒簡介 22
2-5-2 量子粒光學特性 26
2-5-3 生物結合 30
2-6 生物功能化奈米粒子 33
第三章 材料與方法 36
3-1 實驗架構 36
3-2 材料 37
3-2-1 藥品及耗材 37
3-2-2 選用菌株 39
3-2-3 緩衝溶液(PBS)的製備 39
3-3 儀器 40
3-4 純菌培養 40
3-4-1 M. immunogenum 培養液製備 40
3-4-2 M. immunogenum 培養 41
3-4-3 P. aeruginosa 培養液製備 41
3-4-4 P. aeruginosa 培養 41
3-5 菌種保存方法 42
3-5-1 菌種保存 42
3-5-2 固體培養基製作及劃碟 42
3-6 超音波震盪測試 43
3-7 閾值與雜訊測試 44
3-8 生物功能化量子粒 44
3-8-1 生物功能化Carboxyl 量子粒(BCQD) 44
3-8-2 生物功能化Streptavidin量子粒(BSQD) 47
3-9 螢光強度測試 49
3-10  PicoGreen®的最佳濃度測試 49
3-11 改良式免疫螢光法 50
3-11-1 改良式免疫螢光法FAb設計 50
3-11-2 改良式免疫螢光法FAb步驟 50
3-11-3 改良式免疫螢光法Streptavidin量子粒步驟 51
3-12 流式細胞儀分析 51
3-12-1 單一染色訊號區塊選定 52
3-12-2 雙重染色訊號區塊選定 54
3-12-3 專一性及靈敏性分析 56
第四章 結果與討論 58
4-1 最佳超音波震盪效應 58
4-2 閾值的選擇 63
4-3 生物功能化Carboxyl量子粒 65
4-3-1 生物功能化Carboxyl量子粒螢光測試 65
4-3-2 反應時間對生物功能化Carboxyl量子粒標定率的影響 67
4-3-3  PicoGreen®的最佳濃度 69
4-3-4 最佳條件下生物功能化Carboxyl量子粒之標定率 74
4-4 生物功能化Streptavidin量子粒 77
4-4-1  PicoGreen®的最佳濃度 78
4-4-2 反應時間對生物功能化Streptavidin量子粒標定率的影響 81
4-4-3 比較兩種Carboxyl及Streptavidin生物功能化量子粒 83
4-5 改良式免疫螢光法 84
4-5-1 最佳2。Ab濃度測試 84
4-5-2 改良式免疫螢光法FAb之專一性及標定率 87
4-5-3 改良式免疫螢光法Streptavidin量子粒之專一性及標定率 88
4-5-4 比較兩種不同之改良式免疫螢光法FAb及SQD 89
4-6 改良式免疫螢光法與生物功能化量子粒總比較 90
第五章 結論與建議 93
5-1 結論 93
5-2 建議 94
第六章 文獻參考 95
附錄錄 104
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