臺灣博碩士論文加值系統

本論文從物理與化學兩個方向，針對單獨細胞探討新的生醫診斷方法，希望可以達成快速、高效率和準確地檢測細菌的目標：(1)物理：以光學作用力檢測抗甲氧西林金黃色葡萄球菌(MRSA)之抗藥性，利用雷射光鉗抓取單獨球菌，測量其作用力。實驗結果顯示，光鉗作用力與細胞壁厚成正比，由於細胞壁的厚度越大抗藥性越大，所以可藉由測量光鉗作用力得知抗藥性大小，本研究成功地利用光鉗在4~5小時內判別VRSA、VISAh和VISA 抗藥性大小。(2)化學：以生化結合專一性檢測革蘭氏陽性菌，利用雙光子聚合方法製造GFP-AcmA’感測平板，捕捉革蘭氏陽性菌，可直接利用一般光學顯微鏡觀察捕捉結果，最後探討以微流道晶片捕捉革蘭氏陽性菌的可行性，先模擬細菌在流道內流經三維結構感測器的現象，根據模擬結果設計適當形狀的網格狀結構，以期能獲致高效率捕捉。

In this study, we developed the novel technique with fast and high efficiency detection on single cell using physical and chemical methods. In physical method: measurements of optical tweezers forces on Multiple-Resistant Staphylococcus Aureus (MRSA) can be used to detect the bacteria drug resistance by their wall thickness. Our results found the optical forces were directly proportional to wall thickness of bacteria. Because this resistance is associated with the increased growth and reduced autolysis of bacteria cell wall, our experiment has been successfully applied to classify different groups of MRSA, including VSSA (NCTC 10442), VISA (Mu 50), and heto-VISA (Mu 3). In chemical method: species-specific PCR can directly detect the gram-positive bacteria. A 3D pad of GFP-AcmA’proteins was made by two-photon induced laser photochemistry to bind gram-positive bacteria and directly observation by microscopy. For fast and high efficiency detection, we propose a 3D mesh structure in the microfluidic chip. To verify this idea, the flow and particle passing through 3D mesh in microfluidic channel was simulated using mathematical model with different elevation angle of 3D mesh structure, then a 3D Ormocomp mesh with the optimized design by simulation result was fabricated in the microfluidic chip to explore the flow and particle distribution in reality.

第一章 緒論 1
1.1研究動機及背景 1
1.2 研究目的 1
1.2.1 抗藥性細菌檢測 1
1.2.2蛋白質生醫探頭 1
1.3 論文架構 2
第二章 理論與文獻回顧 3
2.1 抗甲氧西林金黃色葡萄球菌抗藥性檢測 3
2.2雷射光鉗(OPTICAL TWEEZERS)微操控 4
2.2.1 光鉗原理 4
2.2.2 光鉗發展 7
2.3 革蘭氏陰/陽性菌檢測 11
2.4 定點照護檢驗(POINT-OF-CARE, POC) 13
2.5 雙光子聚合微製造技術 16
2.5.1 雙光子光致聚合原理 16
2.5.2 TPP發展 20
第三章 材料與方法 23
3.1以雷射光鉗對生物細胞作用力的測量 23
3.1.1雷射光鉗操控系統 23
3.1.2 樣本備製 24
3.1.3 實驗方法 24
3.2以雙光子聚合(TPP)技術製造蛋白質生醫探頭 28
3.2.1 TPP微製造系統 28
3.2.2 模擬軟體(COMSOL)簡介 30
3.2.3 微流道備製 31
3.2.4 樣本備製 34
3.2.5 實驗方法 35
第四章 結果與討論 39
4.1以雷射光鉗對生物細胞作用力的測量 39
4.2以雙光子聚合(TPP)技術製造蛋白質生醫探頭 43
4.2.1 GFP-AcmA’結構之生醫功能測試 43
4.2.2 3D結構化蛋白質探頭應用於微流道晶片 46
第五章 結論 53
5.1 光鉗測量生物細胞作用力分析金黃葡萄球抗藥性 53
5.2 以雙光子聚合技術製造蛋白質生醫探頭 53
參考文獻 54

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