【作者】 李杜娟； 王剑平； 王蓉晖； 李延斌；

【Author】 Dujuan Li~1,Jianping Wang~(1*),Ronghui Wang~2,Yanbin Li~(2,3*) (1.College of Biosystems Engineering and Food Science,Zhejiang University,Hangzhou 310029,China;2.Department of Biological and Agriculture Engineering,and 3 Center of Excellence for Poultry Science,University of Arkansas,Fayetteville,AR 72701,USA

【机构】 浙江大学生物系统工程与食品科学学院； 阿肯色大学生物与农业工程系； 阿肯色大学家禽中心；

【摘要】 高致病性H5N1禽流感病毒作为一种对人类健康存在巨大威胁的病毒,发展建立能够对其进行快速检测的技术已经迫在眉睫。本研究建立了一种基于纳米磁珠信号放大的QCM免疫传感器对H5 N1禽流感病毒进行快速、灵敏和特异性地检测。多克隆抗H5N1病毒表面血凝素抗原(HA)的抗体通过16-巯基十六烷基酸(MHDA)自组装膜固定到QCM金电极表面。H5N1病毒被固定在电极表面的抗体捕捉,引起频率和电阻同时产生变化。通过生物素-链霉亲和素的亲和作用在直径为30 nm的纳米磁珠表面修饰抗-H5抗体,而后,使其和吸附到金电极表面的H5N1病毒反应,进一步放大病毒和抗体结合反应的信号。用环境扫描电镜验证结合到QCM电极表面的H5 N1病毒和纳米磁珠。该QCM免疫传感器能够在两小时内检测出0.0128 HA单位的H5N1禽流感病毒。研究发现:病毒检测的信号和病毒的滴定度相关,当病毒的滴定度在0.128～12.8 HA单位范围时,H5N1的滴定度的对数值和频率响应值之间存在线性关系。在非特异性亚型禽流感病毒的检测中,像H3N2、H2N2、H4N8等,并未发现明显的干扰信号。该免疫传感器对家禽的咽拭子样品进行了检测。研究证明,本研究中建立的基于纳米磁珠信号放大的QCM免疫传感器作为一种可选择的技术,在实际应用中快速、灵敏、特异性地检测H5N1禽流感病毒上具有巨大的潜力。更多还原

【Abstract】 As a potential pandemic threat to human health,there has been an urgent need for rapid detection of the highly pathogenic avian influenza(AI) H5N1 virus.In this study,magnetic nanobeads amplification based quartz crystal microbalance (QCM) immunosensor was developed as a new method and application for AI H5N1 virus detection.Polyclonal antibodies against AI H5N1 virus surface antigen HA(Hemagglutinin) were immobilized on the gold surface of the QCM crystal through self-assembled monolayer(SAM) of 16-mercaptohexadecanoic acid(MHDA).Target H5N1 viruses were then captured by the immobilized antibodies,resulting in a change in the frequency.Magnetic nanobeads(diameter,30 nm) coated with anti-H5 antibodies were used for further amplification of the binding reaction between antibody and antigen(virus).Both bindings of target H5N1 viruses and magnetic nanobeads onto the crystal surface were further confirmed by environmental scanning electron microscopy(ESEM).The QCM immunosensor could detect the H5N1 virus at a titer higher than 0.0128 HA unit within 2 h.The nanobeads amplification resulted in much better detection signal for target virus with lower titers.The response of the antibody -antigen(virus) interaction was shown to be virus titer-dependent,and a linear correlation between the logarithmic number of H5N1 virus titers and frequency shift was found from 0.128 to 12.8 HA unit.No significant interference was observed from non-target subtypes such as AI subtypes H3N2,H2N2,and H4N8.The immunosensor was evaluated using chicken tracheal swab samples.This research demonstrated that the magnetic nanobeads amplification based QCM immunosensor has a great potential to be an alternative method for rapid,sensitive,and specific detection of AI virus H5N1 in agricultural, food,environmental and clinical samples.更多还原