State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China.
Analyst. 2020 Sep 28;145(19):6298-6306. doi: 10.1039/d0an01513j.
An analytical method for screening aptamers for different recognition sites in lactoferrin (Lac) molecules has been developed based on Surface Plasmon Resonance imaging (SPRi), combined with the cluster classification calculation of a quasi-aptamer library strategy and molecular docking simulation analysis. Using the software simulation, a homology analysis was performed on the selected quasi-aptamer sequences, which could be divided into 8 different families. Based on the principle of biomolecular recognition, a label-free, high-throughput dual immune site screening method was established, in which the nucleic acid aptamers of recognizing ability for lactoferrin molecules were fixed onto the surface of the SPRi sensor chip and could bind to the lactoferrin molecules. Then, the aptamer candidates to be paired were introduced, and the recognition event of the second immune site was judged by observing the binding signal of SPRi. The paired SPRi signal was generated only when the site identified by the second nucleic acid molecule was different from the first immune site. Based on this principle, a pair of Lac nucleic acid aptamers (Lac-8 and Lac-25) was finally screened and confirmed using computerized simulation, and has been employed to assay Lac in milk by ELONA (Enzyme-Linked Oligonucleotide Assay).
已开发出一种基于表面等离子体共振成像（SPRi）的分析方法，用于筛选乳铁蛋白（Lac）分子中不同识别位点的适体，该方法结合了拟适体文库策略的聚类分类计算和分子对接模拟分析。使用软件模拟对所选拟适体序列进行同源性分析，可将其分为 8 个不同家族。基于生物分子识别原理，建立了一种无标记、高通量的双免疫位点筛选方法，将具有识别能力的核酸适体固定在 SPRi 传感器芯片表面，使其能够与乳铁蛋白分子结合。然后，引入配对的适体候选物，并通过观察 SPRi 的结合信号来判断第二个免疫位点的识别事件。只有当第二个核酸分子识别的位点与第一个免疫位点不同时，才会产生配对的 SPRi 信号。基于这一原理，通过计算机模拟最终筛选并确认了一对 Lac 核酸适体（Lac-8 和 Lac-25），并已用于通过 ELONA（酶联寡核苷酸测定法）测定乳中的 Lac。
