A.N. Bach Institute of Biochemistry, Research Center of Biotechnology, Russian Academy of Sciences, 119071 Moscow, Russia.
Faculty of Chemistry, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia.
Sensors (Basel). 2024 May 19;24(10):3230. doi: 10.3390/s24103230.
Fluorescence induced by the excitation of a fluorophore with plane-polarized light has a different polarization depending on the size of the fluorophore-containing reagent and the rate of its rotation. Based on this effect, many analytical systems have been implemented in which an analyte contained in a sample and labeled with a fluorophore (usually fluorescein) competes to bind to antibodies. Replacing antibodies in such assays with aptamers, low-cost and stable oligonucleotide receptors, is complicated because binding a fluorophore to them causes a less significant change in the polarization of emissions. This work proposes and characterizes the compounds of the reaction medium that improve analyte binding and reduce the mobility of the aptamer-fluorophore complex, providing a higher analytical signal and a lower detection limit. This study was conducted on aflatoxin B1 (AFB1), a ubiquitous toxicant contaminating foods of plant origins. Eight aptamers specific to AFB1 with the same binding site and different regions stabilizing their structures were compared for affinity, based on which the aptamer with 38 nucleotides in length was selected. The polymers that interact reversibly with oligonucleotides, such as poly-L-lysine and polyethylene glycol, were tested. It was found that they provide the desired reduction in the depolarization of emitted light as well as high concentrations of magnesium cations. In the selected optimal medium, AFB1 detection reached a limit of 1 ng/mL, which was 12 times lower than in the tris buffer commonly used for anti-AFB1 aptamers. The assay time was 30 min. This method is suitable for controlling almond samples according to the maximum permissible levels of their contamination by AFB1. The proposed approach could be applied to improve other aptamer-based analytical systems.
平面偏振光激发荧光团产生的荧光的偏振度取决于包含荧光团的试剂的大小和其旋转速度。基于这一效应，许多分析系统已经被实现，其中样品中包含的分析物与荧光团（通常为荧光素）标记，并与抗体竞争结合。在这些测定中用适配体代替抗体，适配体是低成本且稳定的寡核苷酸受体，这是复杂的，因为将荧光团结合到它们上会导致发射的偏振度发生较小的变化。本工作提出并表征了反应介质中的化合物，这些化合物可以改善分析物的结合并降低适配体-荧光团复合物的迁移率，从而提供更高的分析信号和更低的检测限。本研究针对黄曲霉毒素 B1（AFB1）进行，这是一种普遍存在的有毒物质，污染了植物来源的食品。对 8 种具有相同结合位点但不同区域稳定其结构的 AFB1 特异性适配体进行了亲和力比较，根据这一点，选择了 38 个核苷酸长的适配体。测试了与寡核苷酸可逆相互作用的聚合物，如聚赖氨酸和聚乙二醇。结果发现，它们提供了所需的发射光去偏振度降低，以及高浓度的镁阳离子。在选定的最佳介质中，AFB1 的检测达到了 1ng/mL 的极限，比通常用于抗 AFB1 适配体的 tris 缓冲液低 12 倍。测定时间为 30 分钟。该方法适用于根据杏仁样品中 AFB1 的最大允许污染水平进行控制。所提出的方法可应用于改善其他基于适配体的分析系统。
