基于甘露糖功能化的水凝胶用于E.coli O157：H7的检测

Detection of E.coli O157:H7 Using Mannose-functionalized Hydrogels

利用伴刀豆球蛋白A(Con A)的多价结合能力,结合水凝胶技术与核酸染色技术发展了一种基于甘露糖功能化的水凝胶检测大肠杆菌(E.coli)O157∶H7的方法.以过硫酸铵(APS)为催化剂,四甲基乙二胺(TEMED)为加速剂,用丙烯酰胺(AAm)、N,N-二甲基双丙烯酰胺和N-丙烯酰氧琥珀酰亚胺(NAS)合成水凝胶,通过氨基化甘露糖与NAS发生交联反应,制备了甘露糖功能化的水凝胶.当甘露糖功能化的水凝胶加入与Con A共孵育后的菌悬液中时,由于Con A既能与甘露糖特异性结合,又能与E.coli O157∶H7表面的O-抗原发生免疫反应而紧密连接,使目标菌被捕获到水凝胶表面,采用核酸染料SYBR GreenⅠ对捕获细菌进行染色,实现了对E.coli O157∶H7的核酸标记,最后通过活体荧光成像系统对水凝胶进行荧光成像,从而实现对待测样品的检测.研究结果表明,该方法可应用于缓冲液体系和混合细菌样品中E.coli O157∶H7的特异性检测,且整个检测步骤包括样品预处理可在2 h内完成.该方法成本低、易操作,且具有较好的灵敏度,可检出3.7×101Cells/mL的目标细菌样品.

By combining hydrogel with the nucleic acid dye SYBR Green Ⅰ,we have engineered mannose-functionalized hydrogel for E.coli O157∶ H7 detection based on multivalent binding of Con A.In this method,the hydrogel was first synthesized using acrylamide,N,N-dimethylacrylamide and N-acryloxysuccinimide,employing ammonium persulfate（APS） as the catalyst and etramethylethylenediamine（TEMED） as the accelerator.Then,the mannose was conjugated to the surface of the prepared hydrogel.After preparing the functional hydrogel,the sample was incubated with the hydrogel for 1 h and then stained with a nucleic acid dye SYBR Green Ⅰ.Finally the labeled E.coli O157∶ H7 was determined by the fluorescence in vivo imaging.The detection of E.coli O157∶ H7 was successfully carried out in buffer and bacterial mixture,respectively.This assay allowed the detection of E.coli O157∶ H7 in PB buffer as low as 3.7×101 Cells/mL within 2 h.