集成化酶联免疫微流控平台检测红酒中的赭曲霉毒素A

Determination of ochratoxin A in red wine by integrated enzyme-linked immune microfluidic platform

目的基于微流控芯片在自动化、检测成本上的优势,建立高效的间接竞争酶联免疫吸附法检测红酒中的赭曲霉毒素A(ochratoxin A,OTA)的方法。方法设计按压阀控制的微流控芯片,将含有OTA的样本以及其余4种免疫检测试剂注射进入芯片中。通过负压泵驱动这些试剂分别流经包被有OTA的硅膜夹层进行检测。确定抗原和抗体的工作浓度,绘制拟合标准曲线,计算检出限,分析真实样本的加标回收率。结果抗原为鸡蛋卵白蛋白(ovalbumin,OVA)偶联的OTA,孵育质量浓度为5.0μg/mL,捕获抗体为鼠抗OTA抗体,工作质量浓度为5.0μg/mL,检测抗体为辣根过氧化物酶(horseradish peroxidase,HRP)标记的羊抗小鼠免疫球蛋白G(immunoglobulin G,IgG),稀释倍数为1:1000,OTA的线性范围为1~32 ng/mL,检出限为0.632 ng/mL,干红葡萄酒中的回收率为94.48%~105.76%;相对标准偏差为6.54%~11.18%。结论建立的方法检测成本低,灵敏度高,准确性好,可用于红酒中OTA的定量检测,为检测食品中的生物标志物提供参考。

Objective To establish a method for determination of ochratoxin A(OTA)in red wine by indirect competitive enzyme-linked immunosorbent assay based on the advantages of the automation and cost-efficiency offered by microfluidic chips.Methods The microfluidic chip controlled by the press valve was designed,and samples containing OTA and 4 detection reagents were injected into the chip.These reagents were driven by a negative pressure pump through the OTA coated silicon film interlayer for detection.The working concentrations of antigen and antibody were determined,the fitting standard curve was established,the limit of detection was calculated,and the recovery rate of the real sample was assessed.Results The antigen was OTA conjugating ovalbumin(OVA)at an incubation mass concentration of 5.0μg/mL.The capture antibody was mouse anti-OTA at a working mass concentration of 5.0μg/mL.The detection antibody was sheep anti-mouse immunoglobulin G(IgG)labeled by horseradish peroxidase(HRP)at the dilution ratio of 1:1000.The linear range of OTA was 1–32 ng/mL,achieving the limit of detection was 0.632 ng/mL.The recoveries of OTA in dry red wine were 94.48%–105.76%.The relative standard deviations were 6.54%–11.18%.Conclusion The established method has cost-effectiveness,high sensitivity,and exceptional accuracy,making it well-suited for quantitative OTA detection in wine,providing a reference for the detection of biomarkers in food.