PDF(Pubmed)
Abstract：
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.
摘要：
由平面偏振光激发荧光团引起的荧光具有不同的偏振，这取决于含荧光团试剂的大小及其旋转速率。基于这种效应,已经实施了许多分析系统,其中包含在样品中并且用荧光团(通常是荧光素)标记的分析物竞争结合抗体。用适体代替这种测定中的抗体，低成本和稳定的寡核苷酸受体，是复杂的，因为将荧光团结合到它们会导致发射极化的变化不太明显。这项工作提出并表征了反应介质的化合物，这些化合物可以改善分析物的结合并降低适体-荧光团复合物的迁移率，提供较高的分析信号和较低的检测限。本研究对黄曲霉毒素B1(AFB1)、一种无处不在的有毒物质，污染植物来源的食物。比较了8种具有相同结合位点和不同区域稳定其结构的AFB1特异性适体的亲和力，基于此选择具有38个核苷酸长度的适体。与寡核苷酸可逆相互作用的聚合物，如聚-L-赖氨酸和聚乙二醇，进行了测试。发现它们提供了所需的发射光的去极化减少以及高浓度的镁阳离子。在选定的最佳培养基中，AFB1检测达到1ng/mL的极限，比通常用于抗AFB1适体的tris缓冲液低12倍。测定时间为30分钟。此方法适用于根据AFB1污染的最大允许水平控制杏仁样品。所提出的方法可以应用于改进其他基于适体的分析系统。
