The detection of insulin is an important analytical task. Previously, guanine-rich DNA was believed to bind insulin, and an insulin aptamer was selected based on a few guanine-rich libraries. Insulin is a unique analyte, and it forms different aggregation states as a function of its concentration and buffer conditions, which may affect the detection of insulin. Herein, using fluorescence polarization assays, three insulin preparation methods were evaluated: direct dissolution, ethylenediaminetetraacetic acid (EDTA) treatment to remove Zn²⁺, and dissolution in acid followed by neutralization. All the insulin samples containing Zn²⁺ barely bind to the aptamer DNA, whereas monomers and dimers of insulin with Zn²⁺ removed were able to bind. Compared to the previously reported aptamer, C-rich DNA showed stronger binding affinities and faster binding kinetics. The sigmoidal binding curves and slow binding kinetics showed that multiple DNA strands and insulin molecules gradually bind, and it took approximately 1 h to reach saturation. This insulin binding was nonspecific, and other tested proteins also can bind to C-rich and G-rich DNA with even strong affinities. These results provide important information on the detection of insulin and further insights into the binding mechanisms between oligomeric insulin and DNA.

中文翻译：

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富含胞嘧啶的 DNA 结合胰岛素比富含鸟嘌呤的适体更强：胰岛素聚集对其检测的影响

胰岛素的检测是一项重要的分析任务。以前，富含鸟嘌呤的 DNA 被认为可以结合胰岛素，并且基于一些富含鸟嘌呤的文库选择了胰岛素适体。胰岛素是一种独特的分析物，它会根据其浓度和缓冲条件形成不同的聚集状态，这可能会影响胰岛素的检测。在此，使用荧光偏振测定法，评估了三种胰岛素制备方法：直接溶解、乙二胺四乙酸 (EDTA) 处理以去除 Zn ²⁺以及在酸中溶解然后中和。^{所有含有 Zn 2+}的胰岛素样品几乎不与适体 DNA 结合，而含有 Zn ^{2+的胰岛素单体和二聚体}删除后能够绑定。与之前报道的适体相比，富含 C 的 DNA 显示出更强的结合亲和力和更快的结合动力学。S 形结合曲线和缓慢的结合动力学表明，多条 DNA 链和胰岛素分子逐渐结合，大约需要 1 小时才能达到饱和。这种胰岛素结合是非特异性的，其他测试的蛋白质也可以与富含 C 和 G 的 DNA 结合，甚至具有很强的亲和力。这些结果提供了有关胰岛素检测的重要信息，并进一步深入了解了低聚胰岛素和 DNA 之间的结合机制。