纳米金生物条形码技术检测坚果中的黄曲霉毒素B_1

Detection of AFB_1 in Nuts by a Nanoparticle-based Bio-barcode Assay

利用金纳米颗粒、黄曲霉毒素B1(AFB1)多抗和互补纳米金探针链、条形码DNA链制备纳米金探针(NP),纯化后进行透射电子显微镜(TEM)、紫外-可见光谱(UV-vis)和浓度鉴定。将其与黄曲霉毒素B1单抗修饰的磁性微球探针(MMP)通过抗原抗体作用连接,制备MMP-AFB1-NP三明治复合物结构,利用实时荧光定量PCR(FQ-PCR)检测在高温低盐条件下解链的条形码DNA,通过考察反应结束后每个反应管内的荧光信号到达设定的域值时所经历的循环数值与相应起始模板拷贝数之间的线性相关程度,建立黄曲霉毒素B1的生物条形码检测方法,并对检测体系进行了方法学评价。结果表明,本实验所建立的方法具有快速灵敏、特异性高等特点,每个模板的循环数值与该模板的起始拷贝数的对数具明显的线性关系y=-2.9054x+54.581,r=0.9991,检测灵敏度远远超过酶联免疫吸附法(ELISA)可达10-8 ng/m L,批间批内差值匀小于5%,用建立的生物条形码检测方法(BCA)对结构类似物进行特异性交叉试验,特异性良好。本法可用于花生、腰果等坚果类食品中AFB1的痕量检测。

Gold nanoparticle probes（NP） were prepared using gold（Au） nanoparticles,AFB1 polyclonal antibodies,a complementary gold nanoparticle probe,and barcode DNA,and was characterized after purification using transmission electron microscopy（TEM）,ultraviolet-visible（UV-vis） spectroscopy,and concentration measurement.NPs were coupled with AFB1 monoclonal antibody-modified magnetic microspheres（MMPs） through an antigen-antibody interaction to yield NP-AFB1-MMP sandwich compounds.Fluorescence quantitative-polymerase chain reaction（FQ-PCR） was employed to detect the bio-barcode DNA that was released under high temperature and low salt conditions.A bio-barcode assay for detecting AFB1 was established by investigating the linear correlation between the number of the cycles required for the fluorescence signal in each tube to reach the set threshold value after the reaction,and the number of the corresponding initial template copies,and methodological evaluation was conducted on the detection system.The results indicated that the method established in this experiment is rapid,sensitive,and highly specific.A significant linear relationship was observed between the number of the cycles in each template and the logarithm of the number of initial copies of this template（y=-2.9054x＋54.581,r=0.9991）.The detection sensitivity limit was about 10-8 ng/m L,much more sensitive than the limit of an enzyme-linked immunosorbent assay（ELISA）.The intra-and inter-assay CV（coefficient of variation） values of FQ-PCR were less than 5%,indicating satisfactory accuracy.The established bio-barcode assay（BCA） was used to conduct a crossover experiment on compounds with similar structures,and the results showed a good specificity.This method can be used to determine trace amounts of AFB1 in peanuts,cashews,and other nuts.