免疫竞争比色传感用于测定小麦中呕吐毒素的方法研究

Determination of deoxynivalenol in grain by competitive immunoassay and colorimetric sensor

目的 基于磁分离竞争免疫和酶催化比色传感技术建立一种呕吐毒素快速检测方法。方法 将抗体对呕吐毒素的特异性识别与辣根过氧化物酶催化3,3’,5,5’-四甲基联苯胺/双氧水的比色反应相结合,考察酶催化显色反应的可行性和线性范围,优化反应温度和反应时间。呕吐毒素检测过程集成自动化反应装置和高通量终端检测设备,以呕吐毒素抗体功能化的磁性微粒为固相载体,优化磁珠用量、酶标抗原浓度和免疫亲和反应时间。结果 呕吐毒素在0~1750 ng/mL质量浓度范围内与催化反应产物的吸光度呈良好的线性关系,线性相关系数为0.98。以该方法对小麦基质进行加标回收,回收率在81.3%~90.3%之间,相对标准偏差小于等于2.1%,检测结果经液相色谱-串联质谱法验证相关性良好。结论 本方法具有较高的灵敏度与准确度,且自动化程度较高、操作较为便捷,有潜力应用于粮食样品中呕吐毒素的现场检测。

Objective To establish a rapid detection method of deoxynivalenol based on magnetic-separation competitive immunity and enzyme catalyzed colorimetric sensing technology. Methods The method combines the specific recognition of antibody to deoxynivalenol and the colorimetric reaction of 3,3’,5,5’-tetramethylbenzidine/hydrogen peroxide catalyzed by horseradish peroxidase. The feasibility and linear range of enzyme catalyzed color reaction were investigated, the temperature and time of the catalytic reaction were optimized. The deoxynivalenol detection process integrates an automatic reaction equipment and a high-throughput terminal detection device. With the deoxynivalenol antibody functionalized magnetic beads as the solid carrier, the amount of magnetic beads, the concentration of enzyme-labeled antigen and the reaction time of immune affinity were optimized. Results There was a good linear relationship between deoxynivalenol and the absorbance of catalytic reaction products in the mass concentration range of 0-1750 ng/mL, and the linear correlation coefficient was 0.98. The method was applied for spiked recovery of wheat matrix, and the recoveries were within 81.3%-90.3%, with the relative standard deviation less than or equal to 2.1%. The correlation between the detection results and liquid chromatographytandem mass spectrometry was verified to be good. Conclusion This method has high sensitivity, accuracy, high degree of automation and convenient operation, and has the potential to be applied to the field detection of deoxynivalenol in grain samples.