應用壓阻式微懸臂梁生物感測器偵測巴斯德桿菌之研究

Abstract

近年來台灣的養殖魚業蓬勃發展，而水質檢測一直是其中不可或缺的一環，當魚群處於含菌量高於正常值的海水內，便會開始產生疾病甚至大量死亡，因此如何快速定性與定量檢測海水內之含菌量成為重要的課題。海鱺為台灣高經濟價值之海水養殖魚類之一，其受感染最嚴重之病原體為巴斯德桿菌，感染巴斯德桿菌症之魚群若不及時給予治療，則會引起細菌性敗血症，致死率相當高。前常見之生物感測器大多利用化學或螢光標定的方式來檢測，由於螢光處理複雜，發展免標定病原體之生物感測器是重要方向，本研究利用力學為基礎以微懸臂梁作為生物訊號的轉換機制，透過化學分子(Linker)鍵結專一性抗體與特定病原體，將生物鍵結時對微懸臂梁所產生的表面應力轉換成電訊號輸出，並以後端放大濾波電路處理再輸入電腦做分析。本研究以巴斯德桿菌作為應用對象，實驗結果顯示利用壓阻式微懸臂梁生物感測器檢測數個不同濃度之巴斯德桿菌液，可成功地偵測真實訊號，並以定性與定量地分辨不同濃度之訊號，也透過多次實驗得到每個濃度的訊號範圍(104~108 cfu/ml)，並以掃描式電子顯微鏡的觀察得到巴斯德桿菌鍵結於微懸臂梁上的實證。藉由實驗可知本研究設計之壓阻式微懸臂梁生物感測器的最低量測濃度大約在2×104 cfu/ml，也是目前利用表面應力機制可量的最低濃度，且為目前量測裝置體積最小的系統，並且擁有快速檢測、即時取得反應訊號的優點。未來利用微機電製程來製作壓阻式微懸臂梁生物感測器，可將體積微小化並降低製造成本。

Aquaculture industry has gained prosperous and rapid development in Taiwan recently, in which the monitoring of water quality is an essential part of fish protection. When fish are being bred in seawater with bacteria concentration that is higher than standard value, mortality rises. Therefore, detection of bacteria in seawater with a quantitative, qualitative, and real-time monitoring method becomes an important issue. Cobia is one of high economical value aquaculture fish in Taiwan and is being seriously threaten by Photobacterium damselae subsp. Piscicida(Pdp.). If the infected fish is not being treated in time, it might lead to Pseudotuberculosis and will soon be decreased.resently, most biosensors are using chemical or fluorescence labeling for bacteria detection. For the purpose of label-free sensing, this study combines the mechanical theory and the transduction of bio-signal, and utilizes the surface stress change induced by the recognition of bio-molecule for on-site real time detection. The signal is presented as voltage output and is analyzed in the computer after amplification. This work shows the real reaction of quantitative and qualitative results capable of distinguishing various concentrations of Pdp. in liquid by the piezoresistive microcantilever. The sensing limitation is about 2?104 cfu/ml. Also, the SEM is used to observe the binding state of Pdp. onto the sensing surface of the microcantilever. In comparison to the resonant frequency microcantilever biosensors, the piezoresistive microcantilever biosensors have advantages over rapid detection and on-site real-time detection.