壓電石英晶體免疫感測器在登革熱與鼠疫檢測上之應用研究

Abstract

中文摘要 登革熱病毒（Dengue virus）可藉由病媒蚊感染人體而導致登革熱或者嚴重的出血性登革熱及登革熱休克症候群，致死率可達15%，目前為止尚無有效的藥物或者疫苗可供預防使用，如何早期篩檢就顯得相當重要。鼠疫桿菌（Yersinia pestis）為鼠疫 ( plague ) 之致病原，站在公共衛生預防的角度上，實有必要建立一套快速且有效的檢測方法。壓電石英晶體免疫感測器之檢測原理在於分析物藉由抗體吸附到石英晶體表面上會產生振盪頻率的變化。本論文主要研究壓電石英晶體免疫感測器 ( Quartz crystal immunosensor )應用於快速檢測登革熱病毒與鼠疫桿菌，利用自組裝單層膜技術 ( Self-assembled monolayer, SAM ) 將其對應之單株抗體固定於晶片表面上用來檢測樣品中存在之登革熱病毒NS1蛋白質或鼠疫桿菌F1蛋白質，固定法中以11- Mercaptoundecanoic acid法有較佳之固定效率。應用本研究室陳建源教授發明之自動吸取裝置檢測樣品，可於五分鐘內完成檢測。樣品中NS1與F1蛋白質兩者檢測結果於濃度0.1∼20μg /ml具有良好線性關係，R2 分別為0.9944與0.9889，系統檢測極限可達0.1μg /mL。將樣品以同濃度等體積和人體血清HSA混合檢測，檢測結果於0.1∼20μg/mL具有良好線性關係，陽性反應為干擾值五倍，顯示檢測結果具專一性。檢測結果之相對標準差均在20%以內。另外模擬樣品於人體血清中檢測，必須在稀釋倍數103以上才能有較佳之檢測效果，檢測極限可達0.1μg /mL。本研究綜合氣相與液相檢測之優點，應用自動吸取裝置成功縮短檢測時間且操作步驟簡易，藉此期望能構築具高靈敏度、低成本、可用於快速檢測之免疫型壓電石英晶體生物感測器。

Abstract Dengue virus is one of the most medically important flaviviruses, and it can cause the dengue fever or severe dengue hemorrhagic fever dengue shock syndrome in human being. However, there are no effective drugs or vaccines developed for the treatment or prevention of dengue fever. Yersinia pestis is the pathogen of plague and transmitted by the mouse flea and caused twenty million people death in Europe in history. Although there was no plaque case happened in Taiwan since 1952, it is necessary to develop a rapid and effective detection method for plague. Biosensors are devices that incorporate a biologically active sensing material into a transducer. Piezoelectric immunosensor, which is based on a combination of specific monoclonal antibody recognition and very sensitive quartz crystal microbalance (QCM). According to Sauerbrey equation , the frequency decrease is proportional to the mass change. Antibody immobilization is most important for development of a piezoelectric immunosensor, and the present immobilization methods are mainly based on self-assembled monolayer (SAM). In this study, we developed the quartz crystal immunosensor with anti-NS1 and anti-F1 monoclonal antibodies immobilized by using 11-Mercaptoundecanoic acid ( 11-MUA ) and Cystamine-glutaraldehyde method, and 11-MUA method had better efficiency of antibodies immobilization. The immunosensors can detect NS1 and F1 protein by using automatic adsorption device in a range of 0.1~20 μg/mL within 5 minutes and the result showed high linear relationship ( R2 are 0.9944 and 0.9889 ) with the detection limit at 0.1 μg/mL. The detection of NS1 and F1 protein mixed with HSA in the same concentration with equal volume showed high specificity and linear relationship, and the average relative standard deviation of the detection were less than 20 %. The successive work of this study is to establishing quartz crystal immunosensor of dengue virus and plague with the characteristics of high sensitivity, rapidly detection, low cost and easy operation.