基于荧光生物传感技术检测牛奶中金黄色葡萄球菌肠毒素B

Detection of staphylococcal enterotoxins B in milk based on fluorescence biosensing technique

目的利用纳米金粒子(gold nanoparticles,AuNPs)荧光猝灭性能和核酸适配体的高亲和力,构建一种简便、灵敏的纳米金“Turn-on”型荧光生物传感方法检测牛奶中金黄色葡萄球菌肠毒素B(staphylococcal enterotoxins B,SEB)的方法。方法以AuNPs作为荧光体的能量受体(猝灭剂),荧光素-单链DNA(fluorescein-ssDNA,FAM-ssDNA)作为荧光能量供体,冷冻法制备AuNPs-SEB适配体复合物,基于AuNPsSEB适配体复合物/SEB/FAM-ssDNA的竞争性结合,构建纳米金“Turn-on”型荧光生物传感检测方法。对缓冲体系pH和反应时间等条件进行优化,以牛奶为代表对方法检测性能进行验证。结果在优化好的实验条件(pH 7.5、反应时间15 min和反应温度25℃)下,在10^(-1)~10^(4)ng/mL范围内,荧光强度与SEB质量浓度之间呈现良好的线性关系,其相关系数为0.995,检出限为0.062 ng/mL。应用于牛奶样品中SEB的测定,方法回收率为91.2%~108.0%,相对标准偏差在2.6%~5.2%范围之间。结论该纳米金“Turn-on”型荧光生物传感检测技术具有简便、灵敏和准确等优点,可为食品中污染物的检测提供一种可行的新方法。

Objective To construct a simple and sensitive“Turn-on”fluorescence biosensor based on the good fluorescence quenching of gold nanoparticles(AuNPs)and the high affinity of aptamers,and apply to the detection of staphylococcal enterotoxins B(SEB)in milk samples.Methods AuNPs was used as the energy acceptor of fluorophore and fluorescein-ssDNA(FAM-ssDNA)as the fluorescence energy donor.The AuNPs-SEB aptamer complex was prepared by the freezing method.The AuNPs based“Turn-on”fluorescent biosensing assay was constructed due to the competitive binding of AuNPs-SEB aptamer complex/SEB/FAM-ssDNA.Conditions such as pH of the buffer system and reaction time were optimized.The performance of the proposed method was validated with real milk samples.Results Under the optimized experimental conditions(pH 7.5,reaction time 15 min and reaction temperature 25℃),there was a good linear relationship between fluorescence intensity and SEB mass concentration in the range of 10^(-1)-10^(4)ng/mL,with a correlation coefficient of 0.995 and a limit of detection of 0.062 ng/mL.The method was applied to the determination of SEB in milk samples,and the recoveries were 91.2%-108.0%,and the relative standard deviations were 2.6%-5.2%.Conclusion The AuNPs based“Turn-on”fluorescence biosensor has the advantages such as simplicity,sensitivity,and accuracy,which can provide a feasible new method for the detection of contaminants in food.