Ochratoxin A (OTA), a common mycotoxin, has attracted great concern as many foodstuffs can suffer from OTA contamination; OTA causes harmful effects on human and animals. Rapid and sensitive detection of OTA is demanded in many fields for agricultural product quality, food safety, and health. Aptamer fluorescence polarization/anisotropy (FP/FA) assays integrate advantages of nucleic acid aptamers (e.g., easy preparation, high stability, and low cost) and FP/FA analysis (e.g., high sensitivity, rapidity, simplicity, and robustness). Here, we report the preparation of lissamine rhodamine B labeled OTA and developed competitive aptamer fluorescence anisotropy (FA) assays for OTA with signal-off or signal-on responses by using this fluorescently labeled probe. In the signal-off FA assay, the binding between the fluorescent probe and aptamer gave a large FA signal due to molecular volume increase, and the fluorescent probe was displaced from the aptamer in the presence of OTA target, causing FA to decrease. To further enhance the FA change in the signal-off assay, large-sized streptavidin was conjugated on the aptamer, and this assay allowed for a detection limit of 2.5 nM and a more remarkable FA decrease. Furthermore, we found that the fluorescent probe could interact with Tween 20, which caused the fluorescent probe to show a higher FA value than that of the aptamer–fluorescent probe complex. A signal-on FA assay was achieved in the binding buffer containing 0.1% Tween 20, with a detection limit of 10 nM. Signal-off and signal-on FA methods both were selective and enabled detection of OTA spiked in red wine samples, showing capability for target analysis in complex sample matrix.

中文翻译：

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基于Lissamine Rhodamine B作为O曲毒素A标记的基于荧光各向异性的信号关闭和信号开启适体测定

many曲霉毒素A（OTA）是一种常见的霉菌毒素，由于许多食品都可能受到OTA污染而备受关注。OTA对人类和动物造成有害影响。在农产品质量，食品安全和健康的许多领域中，都需要对OTA进行快速灵敏的检测。适体荧光偏振/各向异性（FP / FA）分析整合了核酸适体的优势（例如，易于制备，高稳定性和低成本）和FP / FA分析（例如，高灵敏度，快速，简单和耐用）。在这里，我们报告用赖氨胺若丹明B标记的OTA的制备，并开发了竞争性适体荧光各向异性（FA）测定法，通过使用该荧光标记探针对OTA进行信号关闭或信号开启响应。在脱信号FA分析中，荧光探针与适体之间的结合由于分子体积的增加而产生了较大的FA信号，并且在OTA靶标存在的情况下荧光探针从适体中移出，导致FA降低。为了进一步增强信号关闭测定中的FA变化，将大型链霉亲和素偶联到适体上，并且该测定允许检测限为2.5 nM，并且FA下降更为显着。此外，我们发现荧光探针可能与Tween 20相互作用，这导致荧光探针显示出比适体-荧光探针复合物更高的FA值。在含有0.1％Tween 20的结合缓冲液中实现了FA信号检测，检测极限为10 nM。信号关闭和信号开启FA方法都是选择性的，能够检测红酒样品中加标的OTA，