基于DNaseⅠ辅助目标物循环信号放大的赭曲霉毒素A检测方法

DNaseⅠ-assisted target recycling signal amplification strategy for ochratoxin A detection

基于酶辅助的目标物循环信号放大及氧化石墨烯包被构建了一种快速、灵敏检测赭曲霉毒素A(ochratoxin A,OTA)的荧光分析新方法。该方法利用聚乙烯基吡咯烷酮(poly-vinylpyrrolidone,PVP)包被氧化石墨烯,防止靶标的非特异性吸附,同时保护适配体免受脱氧核糖核酸酶Ⅰ(deoxyribonucleaseⅠ,DNaseⅠ)的裂解。通过条件优化,发现在氧化石墨烯质量浓度为4μg/mL、PVP物质的量浓度为10 nmol/L、PVP分子量为24000、DNaseⅠ活性为40 U、作用时间为40 min时,OTA的检测性能最佳。在OTA物质的量浓度为25~250 nmol/L范围内,荧光强度与OTA物质的量浓度之间具有良好的线性关系,OTA检测限为9.38 nmol/L(信噪比为3)。经验证,该方法的OTA选择特异性良好,回收率为96.78%~110.97%,标准偏差为1.23%~5.00%。该方法实现了红酒、白咖啡和速溶咖啡等实际样品中OTA的准确检测,同时为食品中相关有害因子的检测提供了新的思路和平台。

A simple and sensitive fluorescent method for ochratoxin A(OTA)detection was developed based on enzyme-assisted target recycling signal amplification in combination with graphene oxide coating.The poly-vinylpyrrolidone(PVP)was used as coating material to prevent the unspecific adsorption between target and graphene oxide.Optimum detection performance for OTA was obtained when the graphene oxide was 4μg/mL,the PVP(molecular weight of 24000)was 10 nmol/L,the activity of DNaseⅠwas 40 U and the reaction time was 40 min.The results showed that the fluorescence intensities displayed a good linear relationship with the OTA concentrations between 25 nmol/L and 250 nmol/L.The limit of detection(LOD)was 9.38 nmol/L at the signal to noise ratio of 3.The OTA selection specificity of the method was good.Its recovery rates were from 96.78%to 110.97%,and the standard deviations were from 1.23%to 5.00%.It has been verified to be useful for detecting OTA in red wine,white coffee and instant coffee,and could be expected to provide a new idea and platform for the detection of other relevant safety hazard factors in food.