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研究生:張聖平
研究生(外文):Sheng-Ping Chang
論文名稱:微型懸臂樑感測器應用於日本腦炎病毒檢測之研究
論文名稱(外文):Detection of Japanese encephalitis virus pathogenesis using microcantilever sensor
指導教授:吳志偉吳志偉引用關係
指導教授(外文):Chih-Wei Wu
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:機械與機電工程學系
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:66
中文關鍵詞:微型懸臂樑感測器水膠日本腦炎病毒細胞固定
外文關鍵詞:MicrocantileverHydrogelJapanese encephalitis virusCell- laden
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台灣為東南亞地區日本腦炎病症之主要疫區之ㄧ,對抗日本腦炎病症目前有效之防疫方式為注射疫苗,但也因為現今使用的疫苗具有副作用之顧慮,使防疫上更為艱辛,故了解日本腦炎對人體致病之感染過程,進而判斷用藥時機及開發新疫苗為刻不容緩之事。常見與病毒相關之檢測分析實驗,皆須於實驗室中之大型儀器進行分析,雖然檢測設備性能精準,但伴隨有所需之檢測時間長,大多需要48小時或以上、待測樣本量多、與無法即時觀測等缺點,導致檢測人力成本的上昇與資源的浪費。提供一低樣本需求之晶片,可快速分析細胞感染病毒之致病過程,並可於感染病毒時即時監測之系統,為本論文研究之目標。
本研究利用微機電製程技術製作微型懸臂樑感測晶片,其檢測晶片大小為3.15 x 3.15 cm,並整合微流道系統、光學量測系統、與具生物相容性之水膠材料,製作一微型生物檢測系統,用以檢測日本腦炎病毒對細胞之感染過程。首先將PDMS微流道系統與製作完成之微型懸臂樑感測晶片利用氧電漿轟擊以進行表面改質並接合,再於微流道系統中通入水膠溶液進行曝光固化,經過沖洗殘餘水膠並進行晶片滅菌之程序後,通入含有細胞之培養液,使細胞貼附於水膠膠塊上生長,最後將日本腦炎病毒通入微流道系統中進行細胞感染,並利用光學偵測系統量測微型懸臂樑之形變量,以此形變量之變化了解日本腦炎病毒對細胞感染使其致病之過程。
本研究成功利用PHEMA水膠將BHK-21細胞固定與培養於微型懸臂樑上,並於細胞固定於水膠膠塊表面完成後通入含有日本腦炎病毒之緩衝液對細胞進行感染,最後利用光學檢測平台擷取微型懸臂樑之形變量變化,由實驗結果得知在細胞受病毒感染4小時40分後,因病毒進入細胞開始複製組裝而使微型懸臂樑產生615nm之形變量,並於此時間點後組裝完成持續脫離細胞。

In Southeast Asia, Taiwan is the major epidemic for the Japanese encephalitis diseases. In the current, vaccine is an effective method to against Japanese encephalitis diseases. But also because of the vaccine is used today have concerns about it side effects, so that disease prevention is more difficult. Therefore, understanding the pathogenesis of Japanese encephalitis virus (JEV) infection processes in the human body, which provide advice to judge the timing of medication and help development of new vaccine.
Common virus detection and analysis experiment, most of all have to use large detection equipment in the laboratory for analysis. Although it has accurate detection performance, but is accompanied by disadvantage of required long detection time, most take 48 hours or even more, wasting a lot quantity of testing sample and can not be monitoring and measurement in real time, this results of increased human cost of detection and waste of resources. In this study, we provide a chip with low sample demand, our system can be analyzed the pathogenesis of cells infected with virus, and also could monitoring virus infection in real time.
This study has produced a microcantilever sensor chip using MEMS technology. The chip size is 3.15 x 3.15 cm, and integrated microfluidic system, optical measurement system, and a biocompatible hydrogel material, producing a micro-biological detection system to detect Japanese encephalitis virus on the cell infection process. First, the PDMS microfluidic system and produced the microcantilever sensor chip using oxygen plasma bombardment for surface modification and bonding, then injected hydrogel solution into microfluidic channel and exposure UV light to curing, after washing the residual hydrogel and sterilization procedures for the chip, which leads to culture medium containing cells, the cells will attached to gel block and adhesion on surface, and finally penetrating the JEV pass microfluidic system for cell infection, and measurement microcantilever sensor deflection by using optical detection system.

摘要 I
Abstract III
目錄 V
表目錄 VIII
圖目錄 IX
第一章 緒論 1
1.1 前言 1
1.2研究動機 2
1.3研究目的 3
1.4文獻回顧 4
1.4.1 微懸臂樑感測器 5
1.4.2 水膠材料 12
1.4.3細胞固定 14
1.5 論文架構簡介 16
第二章 實驗材料與樣本製備 18
2.1實驗材料 18
2.1.1 水膠材料 18
2.1.2 BHK-21細胞 18
2.1.3日本腦炎病毒 19
2.1.4螢光染劑 20
2.1.5 明膠(gelatin) 20
2.2 樣本製備 22
2.2.1 水膠溶液製備 22
2.2.1.1 PHEMA水膠溶液製備 22
2.2.1.2 PEGDA水膠溶液製備 22
2.2.2 水膠材料溶液曝光成型 23
2.2.3 成型後之水膠膠塊處理 23
第三章 微型懸臂樑日本腦炎病毒檢測系統設計與製作 25
3.1 微型懸臂樑日本腦炎病毒檢測系統設計 25
3.1.1 微型懸臂樑設計 25
3.1.2 細胞固定方法設計 27
3.1.3 水膠固定細胞與細胞之培養 28
3.1.4光學平台檢測原理與架設規劃設計 29
3.1.5 病毒感染與微型懸臂樑形變量量測 32
3.2微型懸臂樑日本腦炎病毒檢測系統製作 32
3.2.1微型懸臂樑感測晶片製作 32
3.2.2 水膠材料實驗 35
3.2.2.1水膠材料對細胞毒性分析實驗 35
3.2.2.2 水膠材料微成型實驗 36
3.2.2.3細胞固定及培養實驗 36
3.2.3 光學檢測系統與流體系統建構 37
3.2.3.1光學檢測系統建構 37
3.2.3.2流體系統建構 38
第四章 結果與討論 39
4.1 抗原抗體接合量測實驗與結果 39
4.2 水膠材料細胞毒性分析實驗與結果 41
4.3 水膠培養細胞實驗與結果 43
4.4 水膠微成型性測試實驗與結果 46
4.5 細胞貼附後感染日本腦炎病毒量測實驗與結果 48
第五章 結論與未來展望 52
5.1 結論 52
5.2 未來展望 53
參考文獻 54
附錄 微型懸臂樑生物晶片製程與參數 60

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