基于生物素-亲和素放大的SPR传感器检测大肠杆菌研究

Streptavidin(SA)-Biotin Amplification Strategy Based SPR Sensor forRapid Detection of Escherichia Coli

建立了一种基于表面等离子体共振(SPR)技术的大肠杆菌特异性快速检测方法。采用EDC/NHS将葡聚糖修饰的CM5芯片表面活化,通过亲和素固定生物素标记的二抗(羊抗兔IgG抗体),利用一抗(兔抗大肠杆菌ATCC25922单克隆抗体)和二抗反应,将兔抗大肠杆菌单克隆抗体固定在传感芯片表面。利用一抗和二抗的质量扩增效应和生物素-亲和素的多级放大效应,实现了对低浓度大肠杆菌ATCC25922的快速检测,提高了SPR传感器灵敏度。利用NaOH溶液对芯片再生,可对多个不同浓度样品进行检测,采用相对响应单位(RU)记录数据。本传感芯片对大肠杆菌ATCC25922响应的线性范围为1.5×102 CFU/mL～1.5×107 CFU/mL,检测限为1.5×102 CFU/mL,相关系数r为0.981 5。这种方法简便快捷,有望成为一种在线检测治病菌的有力手段。

A surface plasmon resonance （SPR） biosensor has been established for rapid detection of Escherichia coll. An activation step performed on CM5 chips was carried using EDC/NHS. The biotin-labeled secondard antibody（ goat anti-rabbit IgG）was immobilised onto CM5 chip modified with streptavidin via the biotin-streptavidin interaction. Then, rabbit anti-E, coli ATCC25922 polyclonal antibody （ primary antibody） was immobilized on sensor chip by antibody-antigen recognition. The detectable signal was amplified by the amplification of primary and secondary antibodies and the streptavidin-biotin amplification strategy. All the SPR experiments were performed used a Biacore 3000 and CM5 chips. Regeneration was achieved using NaOH in order to detect several samples. The change of RU was linearly correlated with the concentration of E. coli ATCC25922 （ r = 0. 981 5 ）. This method exhibited high performance with a dynamic range of 1.5 × 102 - 1.5 × 107 CFU/mL, and a detection limit was 1.5×102 CFU/mL. This method has exhibited great potential in bacteriological testing.