Liu Yufei, Yan Huijuan, Shangguan Jingfang, Yang Xue, Wang Meili, Liu Wei
School of Pharmacy, Xinxiang Medical University, Xinxiang, Henan, 453003, People's Republic of China.
Quality monitoring center of agricultural product, Pingdingshan, Henan, 467000, People's Republic of China.
Mikrochim Acta. 2018 Aug 22;185(9):427. doi: 10.1007/s00604-018-2962-8.
A fluorometric aptamer-based assay for ochratoxin A (OTA) is described. It is making use of magnetic separation and a cationic conjugated fluorescent polymer. Amino-tagged aptamer (Apt) against OTA is immobilized on magnetic beads (MBs) to form a conjugate of type Apt-MBs. The immobilized aptamer is partially complementary to carboxyfluorescein-labeled DNA which binds to the Apt-MBs via hybridization if OTA is absent. Only few FAM-DNA will remain in the supernatant after magnetic separation, and only weak fluorescence resonance energy transfer (FRET) occurs on addition of the fluorescent polymer. If, however, OTA is present, it will bind to the aptamer and prevent the hybridization between Apt-DNA and FAM-DNA. This results in the presence of large amounts of FAM-DNA in the supernatant after magnetic separation. On addition of fluorescent polymer, efficient FRET occurs from the polymer to FAM-DNA. Fluorescence, best measured at excitation/emission peaks of 370/530 nm, increases with increasing concentrations of OTA. This assay is highly sensitive and selective. The detection limit is as low as 0.11 ng mL. This is 6 times lower than the aptamer assay without using the fluorescent polymer. Conceivably, this method has a wider scope in that it may be extended to other mycotoxins by simply changing the aptamer. Graphical Abstract Schematic of a fluorometric aptamer assay for ochratoxin A (OTA). It is based on magnetic separation coupled with a cationic conjugated polymer (PFP).
本文描述了一种基于荧光适配体的赭曲霉毒素A（OTA）检测方法。该方法利用磁分离和阳离子共轭荧光聚合物。抗OTA的氨基标记适配体（Apt）固定在磁珠（MBs）上，形成Apt-MBs共轭物。固定化的适配体与羧基荧光素标记的DNA部分互补，若不存在OTA，后者通过杂交与Apt-MBs结合。磁分离后，上清液中仅残留少量FAM-DNA，加入荧光聚合物后仅发生微弱的荧光共振能量转移（FRET）。然而，若存在OTA，它会与适配体结合，阻止Apt-DNA与FAM-DNA杂交。这导致磁分离后上清液中存在大量FAM-DNA。加入荧光聚合物后，聚合物与FAM-DNA之间发生高效FRET。荧光在370/530 nm激发/发射峰处测量最佳，随OTA浓度增加而增强。该检测方法具有高灵敏度和高选择性。检测限低至0.11 ng/mL。这比不使用荧光聚合物的适配体检测方法低6倍。可以想象，该方法具有更广泛的应用范围，因为通过简单更换适配体，它可能扩展到其他霉菌毒素。图形摘要 赭曲霉毒素A（OTA）荧光适配体检测示意图。它基于磁分离与阳离子共轭聚合物（PFP）联用。
