两种常用适配体的纳米金比色法快速检测牛奶中黄曲霉毒素M_(1)的评价研究

Evaluation of Gold Nanoparticles Colorimetric Sensing Based on Two Commonly Aptamer for Rapid Detecting Aflatoxin M_(1) in Milk

目的:建立基于不同序列长度适配体的纳米金(Gold nanoparticles,AuNPs)比色传感法快速定量检测牛奶中的黄曲霉毒素M_(1)(Aflatoxin M_(1),AFM_(1)),并评价目前文献报道中常用的21(A21)和72个碱基(A72)长度的AFM1适配体在实际样品中的检测性能。方法:采用柠檬酸钠还原法制备AuNPs溶液,加入AFM1适配体及AFM1标准品后,适配体与AFM1特异性结合形成特殊三维结构,随着NaCl溶液加入,AuNPs溶液的稳定性被破坏而发生聚集,导致溶液颜色变化,通过测定AuNPs溶液的吸光值和吸收光谱定量检测AFM1。结果:通过优化适配体浓度、NaCl浓度、反应温度及pH等实验条件,基于适配体A21和A72的AuNPs比色传感法检测AFM_(1)的检测限分别为25.26和5.77μg/L,线性范围均为10~800μg/L,且均具有良好的选择性及特异性,在牛奶中的加标回收率分别为95.7%~103.6%、94.3%~98.2%。结论:A72检测牛奶中AFM1的效果优于A21,可能与适配体长度、构型及亲和性相关。基于适配体A72建立的比色传感法简单、快速、灵敏、特异性强且可视化,为AFM1适配体在乳制品中的应用提供数据参考。

Objective:In this study,a colorimetric sensing system based on different aptamers and AuNPs was constructed for the rapid quantitative detection of aflatoxin M_(1)(AFM_(1))in milk.The detection performance of AFM_(1) aptamers with 21(A21)and 72 base(A72)lengths commonly used in literature reports was evaluated in actual samples.Methods:AuNPs solution was prepared by sodium citrate reduction.After adding AFM_(1) aptamer and AFM_(1) standard solution,the aptamer specifically combined with AFM_(1) to form a special three-dimensional structure.With the addition of NaCl solution,the stability of AuNPs solution was disrupted and aggregation occurred,resulting in color change of the solution.AFM_(1) was quantitatively detected by measuring the absorbance value and absorption spectrum of AuNPs solution.Results:Under the optimum condition,the limits of detection of AuNPs-aptasensor based on A21 and A72 aptamers were 25.26µg/L and 5.77µg/L,respectively,with the linear range of 10~800µg/L.Moreover,the AuNPs-aptasensor had good selectivity and specificity.The recoveries of AuNPs-aptasensor based on A21 and A72 aptamers were 95.7%~103.6%and 94.3%~98.2%,respectively.Conclusion:The A72 performed better than A21 in detecting AFM_(1) in milk,which would be related to the length,configuration and affinity of the aptamer.The colorimetric sensing method established based on aptamers A72 was simple,fast,sensitive,highly specific,and visualized,providing data reference for the application of AFM_(1) aptamers in dairy products.