肠致病性大肠埃希菌比色检测的适配体生物传感器研究  

An aptamer-based biosensor for colorimetric detection of Enteropathogenic Escherichia coli

作　　者：吴文鹤[1] 陈勇[1] 蒋璐茜[1] 崔晓阳[1] 聂小博[1] 张杰[1] 吕建新[1] 

机构地区：[1]温州医学院检验医学院遗传学专业,325035

出　　处：《中华检验医学杂志》2010年第7期587-593,共7页Chinese Journal of Laboratory Medicine

基　　金：国家高技术研究发展计划重点资助项目（2007AA022008）;卫生部科学研究基金省部共建计划资助项目（WKJ2008-2-030）;浙江省自然科学基金资助项目（Y2081072）;温州市科技计划重大项目（Y20080080）

摘　　要：目的 结合纳米技术,构建一种可以快速比色检测肠致病性大肠埃希菌的适配体生物传感器.方法 对已有的适配体序列进行截短设计,将截短后的适配体通过酰胺化反应修饰到PDA纳米囊泡的表面,实现生物传感器的组装.利用适配体与LPS间特异性结合和结合后引起PDA纳米囊泡的颜色变化和比色响应值（CR）改变来定性或定量检测溶液中的肠致病性大肠埃希菌.并通过透射电子显微镜对PDA 纳米囊泡与肠致病性大肠埃希菌之间的作用进行验证.结果 成功获得具有结合肠致病性大肠埃希菌功能的适配体截短序列;成功构建可以快速比色检测肠致病性大肠埃希菌的适配体生物传感器,并经透射电镜证实.其不需要特殊仪器,操作简便,检测时间短,仅需30 min;灵敏度较高,检出限为105 CFU/ml,线性范围为105～108CFU/ml;稳定性高,对106 CFU/ml菌液检测的相对标准偏差为6.08%;特异性强,对肠致病性大肠杆菌0111的检测的特异性为100%.结论 本研究成功构建了肠致病性大肠埃希菌适配体生物传感器,实现对肠致病性大肠埃希菌的快速比色检测.Objective To develop and evaluate an aptamer based biosensor （aptasensor） for rapid colorimetric detection of enteropathogenic Escherichia coli （EPEC）. Method The aptasensor was fabricated by modifying the truncated LPS-binding aptamer on the surface of nanoscale polydiacetylene vesicles using peptide bonding between the carboxyl group of the vesicle and the amine group of the aptamer. Molecular recognition between EPEC and aptamer at the interface of the vesicle led to blue-red transition of polydiacetylene which was readily visible to the naked eyes and could be quantified by colorimetric responses （CR）. Transmission electron microscopy （TEM） was used to confirm the specific interactions between EPEC and polydiacetylene vesicles. Result Truncated aptamer showed the similar LPS-binding activity. The aptasensor could detect the target bacteria in a range of 105-108 colony-forming units （CFU）/ml within less than 30 minutes and its specificity was 100% for detection of EPEC O111. The sensor reproducibiliry obtained at 106 CFU/ml was 6. 08% R. S. D. The results of TEM confirmed that the specific interactions between EPEC and polydiacetylene vesicles. Conclusion A new aptasensor was developed successfully for rapid colorimetric detection of EPEC.

关 键 词：肠致病性大肠杆菌 比色法 SELEX适体技术 生物传感技术 

分 类 号：R318.6[医药卫生—生物医学工程]