基于分子发夹/荧光微球的侧向层析法定量检测黄曲霉毒素B1

Determination of Aflatoxin B1 by Lateral Flow Assay Based on Molecule Beacon/Fluorescent Microsphere

基于分子发夹/荧光微球探针构建了一种侧向层析定量检测黄曲霉毒素B1(AFB1)的新方法。一条含有AFB1适配体的分子发夹与荧光微球偶联后,形成的标记探针被喷涂在试纸条的结合垫上。一条5'端含有链霉亲和素的寡核苷酸序列包被在硝酸纤维素膜的检测线上,另一条包含有AFB1适配体互补链的寡核苷酸序列包被在硝酸纤维素膜的质控线上。当待测样本中含有AFB1时,AFB1先与标记物结合垫处标记探针中的AFB1适配体结合,同时,标记探针中的DNA分子发夹‘茎’的双链被打开,AFB1与标记探针形成的复合物层析到反应膜的检测区时,被检测区的寡核苷酸序列捕获,检测区出现亮线。利用该原理,通过一个"off-on"的光信号,实现了对AFB1的高灵敏检测。实验结果表明,AFB1在0.1~50μg/L质量浓度范围内,检测线处的荧光强度(T)和质控线荧光强度(C)的比值与AFB1质量浓度呈良好的线性关系,检出限(S/N=3)达0.05μg/L,且具有很高特异性,可实现对实际样品中AFB1的准确检测。

A new method was proposed for the determination of aflatoxin B1（ AFB1） by lateral flow assay based on molecule beacon（ MB）/fluorescent microsphere（ FM）. A MB containing AFB1 aptamer conjugated with FM,which was coated on the conjugate pad of a strip. A sequence containing adenine（ A） was coated on the nitrocellulose membrane（ NCM） at the test line,and another sequence complementing with AFB1 aptamer was coated on the control line. In the presence of AFB1,the specific combination of AFB1 and aptamer-FM led to the broken off of the double strand in MB.Then,the complex of AFB1/aptamer-FM was captured at the test line,and the excess MB/FM were captured at the control line. The fluorescence was observed with a fluorescence detector,and the content of AFB1 was estimated by the ratio of fluorescence peaks of the test line（ T） and the control line（ C）. The results showed that there existed a good linear relationship between the ratio of fluorescence peaks and AFB1 in the range of 0. 1-50 μg/L with a limit of detection（ S/N = 3） approximately 0. 05 μg/L. With a light signal ＂off-on＂procedure,this proposed method could provide a new way for the accurate determination of AFB1.