基于适配体亲和磁珠的黄曲霉毒素B_1单样本前处理试剂盒的制备及应用

Preparation and Application of Aflatoxin B1 Single Sample Pretreatment Kit Based on Aptamer Affinity Magnetic Beads

黄曲霉毒素B_1(aflatoxin B_1,AFB_1)适配体共价偶联于磁珠表面制备适配体亲和磁珠(aptamer affinity magnetic beads,AAMB),将AAMB、孵育液、洗涤液和洗脱液预封装于试剂条中开发了AFB_1单样本前处理试剂盒,借助真菌毒素全自动净化仪实现了AFB_1前处理的自动化,使用超高效液相色谱仪对洗脱液中AFB_1的含量进行测定。通过优化磁珠粒径、适配体间接臂长度、适配体亲和磁珠用量、孵育液体积确定了AAMB的制备条件以及AFB_1单样本前处理试剂盒的使用条件,对比了手动和自动两种前处理方式下AAMB回收率的差异。在优化条件下,AFB_1的检出限(limit of detection,LOD)为0.6 ng/mL,玉米和小麦中低、中、高三个水平的加标回收率在92.8%~110.1%之间,日内精密度的相对标准偏差(relative standard deviation,RSD)小于5.1%,日间精密度的RSD为6.7%,具有良好的灵敏度、准确性和重复性;粮油基质(玉米、糙米、花生油和花生酱)中AFB_1成分国家有证标准物质的测定结果在其扩展范围内,具有良好的基质适用性。开发的单样本前处理试剂盒借助真菌毒素全自动净化仪仅需一步操作即可在20 min内自动完成10个样品的前处理,具有时间短、通量高、批次间差异小的优点,在真菌毒素定量检测方面具有良好的应用前景,为开发针对其它靶标的自动化前处理方法奠定了基础。

In this study,aflatoxin B1(AFB1)aptamers were covalently coupled on the surface of magnetic beads to prepare aptamer affinity magnetic beads(AAMBs).Furthermore,the AAMBs,incubation solution,washing solution and eluate were pre-encapsulated in reagent strips to develop a single sample pretreatment kit of AFB1.The automatic pretreatment of AFB1 was realized through the automatic mycotoxin purifier.The content of AFB1 in the eluate was determined by ultra high performance liquid chromatography(UPLC).The preparation conditions of AAMB and the use conditions of AFB1 single sample pretreatment kit were determined by optimizing the particle size of magnetic beads,the indirect arm length of aptamer,the amount of AAMB,and the volume of incubation solution.Recoveries was compared by manual and automatic pretreatment methods.Under the optimized conditions,the limit of detection(LOD)was 25 pg/mL,and the spiked recoveries at low,medium and high levels in corn and wheat ranged from 92.8%to 110.1%,with the relative standard deviation(RSD)of intra-day precision less than 5.1%and the RSD of 6.7%in term of inter-day precision,which showed good sensitivity,accuracy and repeatability.The results of AFB1 in the national certified reference materials of grain and oil substrates(corn,brown rice,peanut oil and peanut butter)were within their extended uncertainty range.The single sample pretreatment kit developed could automatically complete the pretreatment of 10 samples within 20 min with the aid of mycotoxin automated purification instrument.This method has the advantages of short time,high throughput,small batch-to-batch variation and no need of cold chain storage and transportation.It has a good application prospect in the quantitative detection of mycotoxins,and could lay a foundation for the development of automatic pretreatment methods for other targets.