基于X型结构的DNA荧光传感器检测黄曲霉毒素B_(1)

DNA fluorescence sensor based on X-shaped structure for detection of AFB_(1)

设计一种X型结构的DNA荧光传感器体系用于高灵敏检测黄曲霉毒素B_(1)(AFB_(1))。在该体系中,用DNA1和DNA2构成了X型骨架并固定在磁珠上,以荧光基团标记的适配体链作为荧光探针(FAM-Apt),根据上清液中荧光信号强度的变化实现对AFB_(1)的快速检测。结果表明:当AFB_(1)不存在时,FAM-Apt可以与X型骨架的4条末端通过碱基互补配对形成双链,经过磁分离后吸取上清液进行检测得到较弱的背景信号;当AFB_(1)存在时,FAM-Apt优先与AFB_(1)结合,游离在上清液中,经过磁分离后得到较强的荧光信号;在最佳试验条件磁珠体积1.5μL、AFB_(1)孵育时间40 min、X型结构中FAM-Apt与X骨架浓度比为4∶1时,传感器的相对荧光强度与lg(ρ_(AFB_(1)))在0.05~100 ng/mL范围内存在良好的线性关系(R^(2)=0.99),检测限低至9 pg/mL。应用所设计的传感器对实际样品进行加标回收测试取得了较为满意的结果。

Aflatoxin B_(1)(AFB_(1))is a typical fungal toxin residual in agricultural products and poses a great threat to both humans and animals when it accumulates to a certain amount,therefore,accurate and rapid determination of AFB_(1)is of great importance for food safety.In this study,an X structure based on DNA nanotechnology was designed and a fluorescent aptamer sensor for the highly sensitive detection of AFB_(1)was constructed by combining magnetic separation technology.In this system,two DNA strands were firstly used to form an X-skeleton and immobilized on magnetic beads,and a fluorescent group-labeled aptamer strand was used as the signal probe(FAM-Apt).When AFB_(1)is not present,FAM-Apt can form a doublestranded structure by base complementary pairing with the four ends of the X-skeleton,followed by magnetic separation and aspiration of the supernatant for fluorescence detection to obtain a low background signal.When AFB_(1)is present,FAM-Apt preferentially binds to AFB_(1)due to the high specificity and affinity of the target and the aptamer,and is detached from the sensor and free in the supernatant,which is detected after magnetic separation to obtain a higher fluorescence signal.Finally,the rapid detection of AFB_(1)was achieved according to the change of fluorescence signal intensity in the supernatant.Under the optimal experimental conditions,the relative fluorescence intensity of the sensor was linearly correlated with lg(ρ_(AFB_(1)))in the range of 0.05-100 ng/mL(R^(2)=0.99),and the detection limit was as low as 9 pg/mL.Satisfactory results were obtained by applying the designed sensor platform to spiked samples for recovery testing.