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研究生： 李建德
Chien-TE LEE
論文名稱： 矽晶表面接枝圖案化聚丙烯酸－電解質多層膜用光學檢測及釋放全血中鼠疫桿菌
Optical Detection of Yersinia Pestis from Whole Blood with Silicon Wafer Surface Grafted Poly(acrylic acid)-Electrolyte Grating and Bacteria Culture after Release.
指導教授： 陳建光
Jem-Kun Chen
口試委員: 蔡協致
Hsieh-Chih Tsai
鄭智嘉
Chih-Chia Cheng
張棋榕
Chi-Jung Chang
學位類別： 碩士
Master
系所名稱： 工程學院 - 材料科學與工程系
Department of Materials Science and Engineering
論文出版年： 2019
畢業學年度： 107
語文別： 中文
論文頁數： 183
中文關鍵詞： 原子自由基轉移聚合法 、聚電解質多層膜 、鼠疫桿菌 、海藻酸 、聚丙烯酸
外文關鍵詞： Atom-transfer radical-polymerization, Layer-by-layer self-assembly, Yersinia Pestis, Poly(acrylic acid), Alginate acid
相關次數： 點閱：330  下載：0 
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    • 本研究為設計一能快速篩檢之高分子材料以偵測人血中的鼠疫桿菌(Yersinia pestis)以及利用聚電解質多層膜的特性進行釋放以利未來更進一步的檢測或試驗，並配合牛血清蛋白的處理並在檢測的同時可不被白血球細胞的干擾。基材為線／間距比率為1：1.5的圖案化光阻矽晶圓表面聚合高分子，使之呈現線寬為1、1.5、2、3μm的一維光柵。以此結構利用原子轉移自由基聚合(Atom Transfer Radical Polymerization, ATRP)製備聚丙烯酸(Poly(acrylic acid)，PAA)高分子刷，進行6、12、18、24小時的聚合成長，可以發現高度隨著時間成長最高來到170nm。接著利用PAA末端的-COOH官能基，透過EDC/NHS的反應接上Protein G與鼠疫桿菌F1抗原之單株抗體(Anti-Yp F1 MoAb)，進行鼠疫桿菌偵測，而此篇利用牛血清蛋白的處理來進行抵抗生物分子的沾黏，達到抗沾黏且可抓取特定細胞的目的。
    檢驗方式利用雷射因光柵繞射產生的能量散失，隨著對於不同的牛血清蛋白比例去測試出在三種比例中1%BSA對WBC在白血球細胞沾黏導致的雷射能量變化量最小。
    接著對於0 CFU/ml -106 CFU/ml做鼠疫桿菌檢體濃度的靈敏度測試，並透過螢光顯微鏡觀察螢光佐證，結果顯示隨著鼠疫檢體濃度的上升，雷射繞射強度呈現下降的趨勢，其靈敏度可達到濃度101 - 102CFU/ml，而螢光顯微鏡已無法發現鼠疫桿菌的螢光存在，說明雷射檢測鼠疫桿菌的靈敏度較螢光顯微鏡來的高。而線寬1μm的圖案化晶片對於不同濃度的鼠疫桿菌有較佳的偵測靈敏度與定量能力，非常有潛力作為即時檢測鼠疫桿菌的技術。
    最後再利用聚電解質多層膜的可脫附性藉由裂解酶來進行細菌釋放，並可以在101 CFU/ml濃度下進行抓取細菌且釋放後可以在培養成功，具有高速檢測及進一步分析的未來性。

    This study designed a rapid screening of polymer materials to detect Yersinia pestis in human blood and the use of polyelectrolyte multilayer membranes for release for further testing or testing in the future, and with bovine serum The protein is processed and not interfered with by white blood cells while being tested. The substrate is a patterned photoresist wafer surface polymer having a line/pitch ratio of 1:1.5, which is a one-dimensional grating having a line width of 1, 1.5, 2, and 3 μm. Using this structure, a polyacrylic acid (PAA) polymer brush was prepared by atom transfer radical polymerization (ATRP), and polymerization was grown up for 6, 12, 18, and 24 hours. The time to grow up to 170nm. Then, using the -COOH functional group at the end of the PAA, a single antibody (Anti-Yp F1 MoAb) of Protein G and Yersinia pestis F1 antigen was ligated through the reaction of EDC/NHS to detect the plaque, and this article utilized bovine serum albumin. The treatment is carried out to resist the adhesion of biomolecules, to achieve anti-adhesion and to grasp specific cells.
    The test method utilizes the energy loss of the laser due to the diffraction of the grating, and the amount of change in the laser energy caused by the adhesion of 1% BSA to WBC in white blood cells in the three ratios is minimized as the ratio of different bovine serum albumin is tested.Then, the sensitivity test of the concentration of the plague bacteria was carried out for 0 CFU/ml -106 CFU/ml, and the fluorescence was confirmed by fluorescence microscope. The results showed that the laser diffraction intensity decreased with the increase of the concentration of the plague specimen. The trend is that the sensitivity can reach a concentration of 101 - 102 CFU / ml, and the fluorescence of the plague bacillus cannot be found by the fluorescence microscope, indicating that the sensitivity of the laser detection of Yersinia pestis is higher than that of the fluorescent microscope. The patterned wafer with a line width of 1 μm has better detection sensitivity and quantitative ability for different concentrations of Yersinia pestis,and has great potential as a technique for detecting Yersinia pestis. Finally, the desorbability of the polyelectrolyte multilayer film is used to release bacteria by lyase, and the bacteria can be grasped at a concentration of 101 CFU/ml and can be successfully cultured after release. The future of high-speed detection and further analysis is possible.

    目錄 指導教授推薦書....................................................................................................I 審定書.................................................................................................................. II 摘要..................................................................................................................... III Abstract ...............................................................................................................V 致謝................................................................................................................... VII 目錄.....................................................................................................................IX 圖目錄..............................................................................................................XIV 1. 緒論................................................................................................................1 1.1 研究背景..............................................................................................1 1.2 研究動機與目的..................................................................................2 2. 理論與文獻回顧............................................................................................3 2.1 高分子刷簡介......................................................................................3 2.2 自組裝單分子層..................................................................................7 2.3 原子轉移自由基聚合法....................................................................10 2.4 液態除氣法........................................................................................14 2.5 智能型高分子....................................................................................16 2.6 微影製程............................................................................................19 2.7 聚電解質多層膜法............................................................................28 2.8 天然高分子........................................................................................31 2.8.1 殼聚醣(Chitosan)..................................................................32 2.8.2 海藻酸(Alginate acid)...........................................................34 2.9 聚丙烯酸(PAA)................................................................................35 2.10 三維結構培養................................................................................36 2.11 細胞釋放........................................................................................38 2.12 細胞培養........................................................................................41 2.13 共價鍵固定法(EDC/NHS Reaction) ..........................................42 2.14 光柵效應........................................................................................43 2.15 重組蛋白與抗體............................................................................46 3. 儀器簡介......................................................................................................47 3.1電漿蝕刻機(Plasma Machine) ..........................................................47 3.2原子力顯微鏡(Atomic Force Microscope, AFM) .............................49 3.3掃描式電子顯微鏡(Scanning Electron Microscope, SEM) .............54 3.4化學分析光譜儀(Electron Spectroscopy for Chemical Analysis, ESCA) ........................................................................................................56 3.5雷射掃描共軛焦顯微鏡(Confocal Laser Scanning Microscope, CLSM) .......................................................................................................59 3.6傅立葉轉換紅外線光譜儀(Fourier-transform infrared spectroscopy, FTIR) .........................................................................................................61 3.7陣列雷射光束分析儀(BeamMic) ......................................................66 3.8接觸角(Contact Angle, CA)...............................................................70 4. 實驗流程與方法..........................................................................................71 4.1實驗流程圖...........................................................................................72 4.2實驗藥品...............................................................................................73 4.3實驗儀器...............................................................................................77 4.4實驗步驟...............................................................................................80 4.4.1微影製程製備圖案化光阻層....................................................80 4.4.2蝕刻製程製備圖案化矽晶圓....................................................82 4.4.3圖案化矽晶片表面起始聚合高分子刷....................................83 4.4.4表面起始ATRP聚合PAA高分子刷.....................................85 4.4.5矽晶片高分子刷表面接枝聚電解質多層膜............................86 4.4.6表面改質ProtrinG與Anti-Yp F1MoAb...............................87 4.4.7白血球抗沾黏實驗....................................................................88 4.4.8細菌抓取、標定、釋放與培養................................................89 5. 結果與討論..................................................................................................92 5.1圖案化表面分析...................................................................................92 5.1.1微影製程光阻圖案....................................................................93 5.1.2線寬型光阻圖案........................................................................93 5.2矽晶片表面高分子刷分析...................................................................96 5.2.1高分子刷表面分析....................................................................96 5.2.2 FT－IR分析...........................................................................100 5.2.3 ESCA光譜分析......................................................................101 5.2.4接觸角親疏水測定..................................................................105 5.3光學性質.............................................................................................107 5.3.1可見光繞射..............................................................................107 5.3.2雷射繞射..................................................................................108 5.3.3高分子雷射偵檢...................................................................... 110 5.3.4雷射細胞偵檢.......................................................................... 116 5.4 接枝聚電解質多層膜分析................................................................ 118 5.5 牛血清蛋白抗白血球沾黏表現........................................................126 5.5.1 白血球抗沾黏測定.................................................................126 5.5.2 牛血清蛋白對雷射偵檢之影響.............................................128 5.6晶片偵測Yersinia pestis之偵測極限分析......................................129 5.6.1 各濃度Yersinia pestis之螢光表現......................................129 5.6.2 全血下各濃度Yersinia Pestis之螢光表現..........................131 5.6.3 各濃度Yersinia Pestis之雷射偵檢之表現..........................134 5.6.4 全血下白血球雷射偵檢探討.................................................136 5.7細胞釋放及培養.................................................................................138 5.7.1鼠疫桿菌的釋放......................................................................139 5.7.2大腸桿菌的抓取與釋放..........................................................143 5.7.3大腸桿菌的培養......................................................................146 6. 結論............................................................................................................148 參考文獻...........................................................................................................149

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