交流電動力輔助之阻抗式生醫感測研究

Abstract

食物中毒案例於全世界的發生率愈來愈高且也愈來愈受重視，其原因主要都是細菌對食物的汙染。在多數有害細菌之中，有病原性的腸炎弧菌在台灣及美國所引起的病發案例最為多數。傳統和現今應用於感測細菌的方式通常需要數天的時間來完成。相較於發展中的生物感測器偵測病原，則可大大地縮短偵測時間。本研究利用電阻抗方式感測貼附於金電極表面的腸炎弧菌，然而為了縮短腸炎弧菌貼附於金電極表面的時間，使用在微流道系統施加不均勻電場所產生的交流電動力濃縮細菌。本研究之生醫感測元件以半導體元件製程製作金電極於二氧化矽基材上。新的電極設計利用圓形電極裡的內圓電極換成指叉式型式。此設計是為了同時具備聚集細菌及感測細菌之效果。為了使聚集的細菌固定在晶片表面，對金電極修飾有專一性的抗體使其與腸炎弧菌有共價鍵連接。我們也利用氧化還原、螢光修飾及TMP 的顯色機制確認晶片上所修飾的化學分子和生物分子。檢測細菌的晶片會與微流道系統作結合，在輸送細菌於晶片表面的同時，施加適當的電壓及頻率使溶液產生交流電動力以聚集細菌置電極內圓。然後量測細菌貼附於電極表面後的阻抗，再與細菌貼附於晶片之前的阻抗值比較，計算出變化量。本研究以不同大小電極檢測腸炎弧菌，發現當電極面積對流道腔體體積之比率愈大，其阻值變化量愈大。對不同濃度的感測，本系統的最低極限為105cfu/mL。最後以量出的阻抗值對一等效電路作模擬，分析電路裡造成阻值改變的元件。等效電路的模擬結果可看出，細菌貼附於晶片表面主要導致表面電容的改變。另外，選擇性感測也呈現在此研究中，當大腸桿菌與腸炎弧菌同時混入溶液一起感測時，其阻值變化量接近單測腸炎弧菌之變化量。

Foodborne disease is becoming one of the major crises on human health. Vibrio parahaemolyticus, one of the pathogens, is becoming a highest risk source in food poisoning worldwide. The conventional bacterial detection methods such as immunology-based methods are not fast enough and require a few days for a reply. Hence, biosensing techniques with rapid, sensitive, and accurate detection have attracted much attention recently. Among different sensing techniques, impedimetric sensor adopted in this study is becoming popular due to its simplicity in device preparation. In this thesis, study of Vibrio parahaemolyticus trapping mechanisms on the gold electrode via AC electrokinetics was investigated. The detection was fulfilled using impedance analysis in a microfluidic system. A novel configuration of electrode was designed and proposed for its advantages in combining bacteria trapping and detection. To enhance the specificity, Vibrio parahaemolyticus specific antibody was modified onto the gold electrode for bacteria immobilization. Cyclic voltammetry, fluorescence imaging, and TMP were applied to identify the quality of the surface modifications. The functionalized chip was then integrated into microfluidic system for bacteria transport. The time needed for bacteria immobilization was reduced by AC electrokinetics manipulation. The concentrated bacteria were detected by impedance analysis. An electrical circuit model was also constructed and compared with the experimental data. In spite that the detection was only at a level of 105cfu/mL, according to analysis, device with larger ratio of electrode surface to volume of microfluidic channel could enhance sensitivity. In addition, the selectivity was also investigated by simultaneous detection of analyte with Vibrio parahaemolyticus and non-pathogenic bacteria, X1 Blue E.coli.