纳米花结合G-四链体对大肠杆菌的可视化检测方法的建立

Establishment of a visual detection method for nanoflowers combined with G-quadruplex for foodborne pathogens

为建立一种新型、快速、可视化的病原菌检测方法,使用刀豆蛋白(ConA)-磷酸铜纳米花结合G-四链体对大肠杆菌(Escherichia coli,E.coli)进行可视化检测,通过纳米花中包含的铜离子(Cu2+)催化水解溶液中叠氮和G-四链体片段之间的点击化学反应,最终形成完整G-四链体结构,简化传统检测方法中的固定及洗涤等步骤。在血红素(hemin)的作用下,激活DNAzyme,使其具有类似辣根过氧化物酶(HRP)的活性;利用四氮甲基联苯胺(TMB)作为底物产生肉眼可视的颜色变化,根据其颜色深浅可对靶标细菌的含量进行定性及定量的分析。结果表明,经过各种条件优化试验及灵敏性、特异性试验得出该病原菌纳米花结合G-四链体可视化检测方法稳定、快速、灵敏,且可以应用于多种病原的检测。

In order to develop a new,rapid and visual method for detecting pathogenic bacteria,concanavalin(ConA)-copper phosphate nanoflowers combined with G-quadruplex were used to visualize E.coli,and the azide and G-quadruplex in the hydrolysis solution were catalyzed by copper ions(Cu2+) contained in nanoflowers.The click chemical reaction between the fragments eventually forms a complete G-quadruplex structure,simplifying the steps of fixation and washing in the conventional detection method.Under the action of hemin,the DNAzyme is activated to have a horseradish peroxidase(HRP)-like activity,and tetrazomethylbenzidine(TMB) is used as a substrate to produce a visually visible color change.Qualitative and quantitative analysis of the target bacteria content based on its color depth.After various conditions optimization experiments and sensitivity and specificity tests,the detection method of the pathogen is stable,rapid,sensitive,and can be applied to the detection of a wide range of bacteria.