量子点荧光微球免疫法定量检测小麦中黄曲霉毒素B1

Quantitative determination of aflatoxin B 1 in wheat by quantum dot fluorescence microsphere immunoassay

目的建立量子点荧光微球免疫法快速检测小麦中黄曲霉毒素B1的方法。方法采用量子点荧光微球作为荧光标记物,与黄曲霉毒素B1的单克隆抗体偶联,构建量子点荧光微球探针。优化缓冲液pH、抗体最小标记量、荧光探针用量和包被抗原浓度等实验条件,建立检测卡上T线和C线信号峰值面积的比值与样本中黄曲霉毒素B1浓度的关系,构建定量标准曲线。针对小麦样品,将该检测方法与时间分辨荧光定量检测方法进行比较。结果本研究建立的荧光定量免疫层析检测方法最佳反应条件为:pH 7.5磷酸钠缓冲液,抗体标记量为20μg,荧光探针用量为4.0μL,抗原质量浓度使用0.40mg/mL。小麦中黄曲霉毒素B1的定量检测线性范围为0.05~25.00μg/kg,相关系数(r^(2))为0.9994,检出限为0.02μg/kg,定量限为0.05μg/kg。加标回收率为91.50%~115.00%,变异系数为1.88%~5.46%。结论本研究建立的荧光定量免疫层析方法快速、准确、稳定性好、可靠性高,适用于小麦中黄曲霉毒素B1的现场快速检测。

Objective To establish a method for rapid detection of aflatoxin B 1 in wheat by quantum dot fluorescent microsphere immunoassay.Methods Quantum dot fluorescent microspheres were constructed as a fluorescent marker coupled to a monoclonal antibody to aflatoxin B 1.Experimental conditions of buffer pH,the minimum amount of antibody labeling,the amount of fluorescent probe used,and the concentration of coated antigen were optimized to establish the relationship between the ratio of the peak area of T line and C line signal on the detection card and the concentration of aflatoxin B1 in the sample,and construct a quantitative standard curve.For the wheat samples,this assay was compared with the time-resolved fluorescence immunoassay.Results The best reaction conditions for fluorescence quantitative immunochromatography established in this study were pH 7.5 sodium phosphate buffer,antibody labeling of 20μg,fluorescent probe of 4.0μL,and antigen mass concentration of 0.40 mg/mL.The linear range of quantitative detection of aflatoxin B1 in wheat was 0.05‒25.00μg/kg,and the correlation coefficient(r^(2))was 0.9994,with the limit of detection of 0.02μg/kg and the limit of quantification of 0.05μg/kg.The addition recoveries ranged from 91.50%to 115.00%,and the coefficients of variation ranged from 1.88%to 5.46%.Conclusion The fluorescence quantitative immunochromatography method established in this study is rapid,accurate,stable and reliable,and is suitable for the rapid detection of aflatoxin B 1 in wheat.