Tang Juan, Huang Yapei, Liu Huiqiong, Zhang Cengceng, Tang Dianping
Key Laboratory of Functional Small Organic Molecule, Ministry of Education, College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang, 330022, China.
Key Laboratory of Analysis and Detection for Food Safety (Ministry of Education & Fujian Province), Institute of Nanomedicine and Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou, 350108, China.
Anal Bioanal Chem. 2016 Dec;408(30):8593-8601. doi: 10.1007/s00216-016-9343-0. Epub 2016 Mar 9.
A new homogeneous electrochemical immunosensing platform was designed for sensitive detection of aflatoxin B (AFB) in foodstuff. The system consisted of anti-AFB antibody labeled DNA (Ab-DNA), AFB-bovine serum albumin (BSA)-conjugated DNA (AFB-DNA), and methylene blue functionalized hairpin DNA. Owing to a specific antigen-antibody reaction between anti-AFB and AFB-BSA, the immunocomplex formed assisted the proximity hybridization of DNA with DNA, thus resulting in the formation of an omega-like DNA junction. Thereafter, the junction opened the hairpin DNA to construct a new double-stranded DNA, which could be readily cleaved by exonuclease III to release the omega-like DNA junction and methylene blue. The dissociated DNA junction could repeatedly hybridize with residual hairpin DNA molecules with exonuclease III-based isothermal cycling amplification, thereby releasing numerous free methylene blue molecules into the detection solution. The as-produced free methylene blue molecules could be captured by a negatively charged indium tin oxide electrode, each of which could produce an electronic signal within the applied potentials. On introduction of target AFB, the analyte competed with AFB-DNA for the conjugated anti-AFB on the Ab-DNA, subsequently decreasing the amount of omega-like DNA junctions formed, hence causing methylene blue labeled hairpin DNA to move far away from the electrode surface. Under optimal conditions the detectable electrochemical signal decreased with increasing amount of target AFB in a dynamic working range of 0.01-30 ng mL with a detection limit of 4.8 pg mL. In addition, the precision and reproducibility of this system were acceptable. Finally, the method was further evaluated for analysis of naturally contaminated or AFB-spiked peanut samples, giving results that matched well with those obtained with a commercial AFB ELISA kit.
设计了一种新型的均相电化学免疫传感平台，用于灵敏检测食品中的黄曲霉毒素B（AFB）。该系统由抗AFB抗体标记的DNA（Ab-DNA）、AFB-牛血清白蛋白（BSA）偶联的DNA（AFB-DNA）和亚甲基蓝功能化的发夹DNA组成。由于抗AFB与AFB-BSA之间存在特异性抗原-抗体反应，形成的免疫复合物有助于DNA与DNA的邻近杂交，从而导致形成ω样DNA连接体。此后，该连接体打开发夹DNA以构建新的双链DNA，其可被核酸外切酶III轻易切割以释放ω样DNA连接体和亚甲基蓝。解离的DNA连接体可通过基于核酸外切酶III的等温循环扩增与残留的发夹DNA分子反复杂交，从而将大量游离的亚甲基蓝分子释放到检测溶液中。产生的游离亚甲基蓝分子可被带负电荷的氧化铟锡电极捕获，每个分子在施加的电位范围内均可产生电子信号。引入目标AFB后，分析物与AFB-DNA竞争Ab-DNA上偶联的抗AFB，随后减少形成的ω样DNA连接体的数量，从而导致亚甲基蓝标记的发夹DNA远离电极表面。在最佳条件下，可检测的电化学信号随目标AFB量的增加而降低，动态工作范围为0.01 - 30 ng/mL，检测限为4.8 pg/mL。此外，该系统的精密度和重现性是可接受的。最后，对该方法进一步评估用于分析天然污染或AFB加标的花生样品，结果与使用商业AFB ELISA试剂盒获得的结果匹配良好。
