臺灣博碩士論文加值系統

細菌黏附到宿主細胞表面是傳染病傳染過程中的第一步驟，因此，比較細菌對宿主細胞的黏附力，是研究黏附作用的必要工作，此一成果將來是用來研發有效的治療藥物或疫苗的重要基礎。為此，本論文希望發展雷射鑷夾成為一種量測黏附力的有效工具。近十餘年來，雷射鑷夾的梯度力日益廣泛被用來捕捉、搬運與操縱單一微小生物體。我們利用傳統水流黏滯力法，校正出雷射鑷夾最大捕捉力和雷射光功率的關係，然後根據恰好能分離細菌與宿主的雷射光功率，可推算所對應的雷射鑷夾最大捕捉力，在力平衡時，雷射鑷夾最大捕捉力即為細菌與宿主之間的黏附力。
本實驗我們使用第五型膠原蛋白代替宿主細胞，再利用雷射鑷夾，分別量測並比較克雷白氏肺炎桿菌珠、移除功能性mrk A纖毛蛋白的突變株、以及還原株（將功能性mrk A纖毛植回突變株）對膠原蛋白的黏附力，比較黏附力強弱。實驗結果顯示，黏附力的強弱依次為：「還原株」、「正常株」、「突變株」，不過其中「還原株」與「正常株」的黏附力接近。雷射鑷夾量測的實驗結果，與傳統塗盤數落菌數的實驗結果趨勢吻合，這證實了「功能性mrk A纖毛」，確實是克雷白氏肺炎桿菌黏附人體的主要介質。

Adherence to host cells by a bacterial pathogen is a critical step for establishment of infection. In order to study the mechanism of adherence, it is necessary to measure the biological force due to adherence. Consequently, optical tweezers become an effective tool for this purpose. In the past decade, the gradient force of optical tweezers has been widely applied in the manipulation of a single small bio object. In this thesis, we use an optical tweezers system to separate a single Klebsiella Pneumoniae, the bacterium, from collagen, the host.
We first increase the laser power of the optical tweezers until the bacterium is detached from the collagen coated on the surface of a polystyrene bead. Then, we obtain the magnitude of the adhesive force between the Klebsiella Pneumoniae and the collagen, which is equal to that of the corresponding trapping force provide by the optical tweezers at that specific laser power. Note that we calibrate the relationship between the trapping force of the optical tweezers and the power of the laser by using the water-drag-force method.
In this experiment, we use Klebsiella Pneumoniae CG43-S3, Klebsiella Pneumoniae CG43-S3-mrk A, and Klebsiella Pneumoniae CG43-S3-mrk A[pYJ01] as the bacterial pathogen, separately. We found that the adhesive force between Klebsiella Pneumoniae CG43-S3 and collagen is comparable with that between Klebsiella Pneumoniae CG43-S3-mrk A[pYJ01] and collagen. And these two forces are apparently stronger than the adhesive force between Klebsiella Pneumoniae CG43-S3-mrk A and collagen. This result by using optical tweezers agrees with that by using the traditional biology method of plating. Our work proves that the functional mrk A cilium was a key factor that enhances the adherence of Klebsiella Pneumoniae to human body

目 錄
中文摘要 LLLLLLLLLLLLLLLLLLLLLLLL Ⅰ
英文摘要 LLLLLLLLLLLLLLLLLLLLLLLL Ⅱ
誌謝 LLLLLLLLLLLLLLLLLLLLLLLL Ⅳ
目錄 LLLLLLLLLLLLLLLLLLLLLLLL Ⅴ
圖目錄 LLLLLLLLLLLLLLLLLLLLLLLL Ⅶ
表目錄 LLLLLLLLLLLLLLLLLLLLLLLL Ⅸ
一 介紹 LLLLLLLLLLLLLLLLLLLLLL 1
1.1 雷射鑷夾之發展背景與特色 LLLLLLLLLLLL 1
1.2 研究動機與目標 LLLLLLLLLLLLLLLLL 2
1.3 論文各章節簡介 LLLLLLLLLLLLLLLLL 3
二 原理 LLLLLLLLLLLLLLLLLLLLLL 4
2.1 雷射鑷夾的捕捉機制 LLLLLLLLLLLLLLL 4
2.2 雷射鑷夾作用力的分析 LLLLLLLLLLLLLL 6
2.3 克雷白式肺炎桿菌纖毛與膠原蛋白簡介 LLLLLLL 12
三 原理與方法 LLLLLLLLLLLLLLLLLLL 14
3.1 雷射鑷夾捕捉力的特性 LLLLLLLLLLLLLL 14
3.1.1 光學彈簧 LLLLLLLLLLLLLLLLLLLL 14
3.1.2 水流黏滯力 LLLLLLLLLLLLLLLLLLL 15
3.1.3 雷射鑷夾捕捉力與微粒子位移關係的校正 LLLLLL 15
3.1.4 雷射鑷夾最大捕捉力與雷射功率關係的校正 LLLLL 17
3.2 實驗裝置與影像分析系統 LLLLLLLLLLLLL 18
3.2.1 實驗裝置 LLLLLLLLLLLLLLLLLLLL 18
3.2.2 影像分析系統 LLLLLLLLLLLLLLLLLL 19
3.3 實驗步驟 LLLLLLLLLLLLLLLLLLLL 20
3.3.1 雷射鑷夾捕捉力特性校正 LLLLLLLLLLLLL 20
3.3.2 生物樣品準備 LLLLLLLLLLLLLLLLLL 21
3.3.3 塗盤數菌落數法 LLLLLLLLLLLLLLLLL 22
3.3.4 雷射鑷夾量測法 LLLLLLLLLLLLLLLLL 23
3.4 雷射鑷夾捕捉力修正 LLLLLLLLLLLLLLL 24
四 實驗結果與討論 LLLLLLLLLLLLLLLLL 25
4.1 雷射鑷夾捕捉力校正 LLLLLLLLLLLLLLL 25
4.1.1 樣品平台移動速度與驅動電壓關係的校正 LLLLLL 25
4.1.2 雷射鑷夾最大捕捉力與雷射功率關係的校正 LLLLL 26
4.2 生物力的量測 LLLLLLLLLLLLLLLLLL 29
4.2.1 雷射鑷夾最大捕捉力與雷射功率關係的校正 LLLLL 29
4.2.2 塗盤數菌落數法 LLLLLLLLLLLLLLLLL 31
4.2.3 雷射鑷夾量測法 LLLLLLLLLLLLLLLLL 32
五 結論 LLLLLLLLLLLLLLLLLLLLLL 35
參考文獻 LLLLLLLLLLLLLLLLLLLLLLLL 37
圖目錄
圖2-1 雷射鑷夾捕捉微粒子的工作機制 LLLLLLLLLLLL 5
圖2-2 EM Model 示意圖 LLLLLLLLLLLLLLLLLLL5
圖2-3 EM Model 之雷射鑷夾橫向力沿Y軸方向的分布LLLLLL 8
圖2-4 細光束入射透明球體示意圖LLLLLLLLLLLLLLL 9
圖2-5 RO Model之雷射鑷夾橫向力沿Y軸方向的分布LLLLLL 11
圖3-1 雷射鑷夾捕捉力與微粒位移關係理論圖LLLLLLLLLLL 14
圖3-2 利用微粒子飄移位移校正雷射鑷夾橫向捕捉LLLLLLLL16
圖3-3 被捕捉之微粒子受水流影響而產生微小的位移LLLLLLL16
圖3-4 雷射鑷夾與影像分析系統的實驗裝置圖LLLLLLLLLL18
圖3-5 利用Labview軟體及Imaq所寫出的「影像分析處理系統」之
操作面板 LLLLLLLLLLLLLLLLLLLLLL 19
圖3-6 克雷白氏肺炎桿菌黏附第五型膠原蛋白之菌落圖案 LLLL 22
圖3-7 將微粒子固定在載玻片上，利用雷射鑷夾捕捉並搬運細菌去黏
微粒子後再移開 LLLLLLLLLLLLLLLLLLL 23
圖3-8 (a)沿著長軸方向流動 (b)沿著短軸方向流動 橢球體受水流
黏滯力示意圖 LLLLLLLLLLLLLLLLLLLL 24
圖4-1 平台的平均移動速度與步徑馬達驅動電壓的關係圖LLLLL26
圖4-2 平台驅動電壓與雷射光功率的關係圖，樣品直徑為1μm的微
粒 LLLLLLLLLLLLLLLLLLLLLLLLL 27
圖4-3 雷射鑷夾最大捕捉力與雷射光功率的關係圖，樣品為直徑為
1μm的微粒子LLLLLLLLLLLLLLLLLLLL 27
圖4-4 平台驅動電壓與雷射光功率的關係圖，樣品為克雷白式肺炎
桿菌LLLLLLLLLLLLLLLLLLLLLLLLL 29
圖4-5 雷射鑷夾最大捕捉力與雷射光功率的關係圖，樣品為克雷
白式肺炎桿菌 LLLLLLLLLLLLLLLLLLLL30
圖4-6 平台驅動電壓與雷射光功率的關係圖，樣品為克雷白式肺炎
桿菌、1μm的微粒子LLLLLLLLLLLLLLLLL30
表目錄
表 2-1 EM Model之模擬參數LLLLLLLLLLLLLLLLL 8
表 2-2 RO Model之模擬參數 LLLLLLLLLLLLLLLLL 11

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