State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China.
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China; National Engineering Research Center for Functional Food, Jiangnan University, Wuxi, 214122, China.
Talanta. 2023 Aug 1;260:124530. doi: 10.1016/j.talanta.2023.124530. Epub 2023 Apr 14.
Aptasensors being versatile sensing platforms presented higher sensitivity toward target detection. However, lacking theoretical basis of recognition between most targets and their corresponding aptamers has impeded their applications. Herein, we conducted a study to explore the binding mechanism of aptamer to kanamycin (Kana) and developed rapid fluorescent aptasensing methods. Based on the fluorescence polarization results, base mutations were performed at different sites of the aptamer. The key binding nucleotides of Kana was identified as T7, T8, C13 and A15 by using isothermal titration calorimetry (ITC). The Kmut3 (2.18 μM) with lower dissociation constants (Kd), one-third of the native aptamer (6.91 μM), was also obtained. In addition, the lower K concentration and temperature were found to be conducive to Kana binding. Circular dichroism (CD) results revealed that the binding of Kana can trigger the change of base stacking force and helix force. On the aforementioned basis, a fluorescent sensor was designed with the native aptamer and Kmut3 as recognition elements. The comparison results proved that the Kmut3 presented a 3 times lower limit of detection of 59 nM compared to the native aptamer (148 nM). Notably, this developed aptasensor can be finished in 45 min and was convenient to operate.
适配体传感器作为多功能传感平台，对目标检测具有更高的灵敏度。然而，大多数目标与其相应适配体之间缺乏识别的理论基础，这阻碍了它们的应用。在此，我们研究了适配体与卡那霉素（Kana）的结合机制，并开发了快速荧光适配体传感方法。基于荧光偏振结果，在适配体的不同位点进行碱基突变。通过等温滴定量热法（ITC）鉴定了 Kana 的关键结合核苷酸为 T7、T8、C13 和 A15。还获得了解离常数（Kd）更低的 Kmut3（2.18 μM），为天然适配体（6.91 μM）的三分之一。此外，较低的 K 浓度和温度有利于 Kana 结合。圆二色性（CD）结果表明，Kana 的结合可以引发碱基堆积力和螺旋力的变化。在此基础上，以天然适配体和 Kmut3 为识别元件设计了荧光传感器。比较结果表明，与天然适配体（148 nM）相比，Kmut3 的检测下限低 3 倍，为 59 nM。值得注意的是，这种开发的适配体传感器可以在 45 分钟内完成，操作方便。
