Zhang Shikun, Ning Zhiyuan, Zhang Yingming, Lin Xianfeng, Duan Nuo, Wang Zhouping, Wu Shijia
State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, 214122, China; School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China.
State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, 214122, China; School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China; International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, 214122, China.
Biosens Bioelectron. 2025 Mar 1;271:117038. doi: 10.1016/j.bios.2024.117038. Epub 2024 Dec 5.
Aptamers, as a kind of recognition molecules with stable nature and excellent binding ability, are usually obtained by systematic evolution of ligands by exponential enrichment (SELEX). However, the traditional SELEX suffers from the problems of low screening efficiency as well as excessive number of screening rounds, making the screening a cumbersome process, which greatly restricts the application and development of aptamers. Here, a microfluidic SELEX platform based on capture SELEX was designed and developed to make the screening more integrated and convenient. Nε-carboxymethyl lysine (CML) and Nε-carboxyethyl lysine (CEL), were selected as targets for screening, and candidate aptamers were identified after eight rounds of screening using the microfluidic SELEX platform. Following isothermal titration calorimetry (ITC) and SYBR GREEN I (SGI) analysis, aptamer S2 was identified with the highest affinity and specificity. Aptamer S2 was further optimized based on the binding sites explored by molecular docking. Eventually, the truncated aptamer S2-40 was obtained, which was superior to S2 in terms of affinity and specificity with dissociation constant (Kd) of 6.65 ± 3.07 μM (ITC) and 42.1 ± 9.34 nM (SGI), respectively. This indicated that the microfluidic SELEX platform offers a more integrated and convenient approach to aptamer screening.
适体作为一类性质稳定且结合能力出色的识别分子，通常通过指数富集配体系统进化技术（SELEX）获得。然而，传统的SELEX存在筛选效率低以及筛选轮次过多的问题，使得筛选过程繁琐，极大地限制了适体的应用与发展。在此，设计并开发了一种基于捕获SELEX的微流控SELEX平台，以使筛选更加集成化和便捷。选择Nε-羧甲基赖氨酸（CML）和Nε-羧乙基赖氨酸（CEL）作为筛选靶点，并使用该微流控SELEX平台经过八轮筛选鉴定出候选适体。通过等温滴定量热法（ITC）和SYBR GREEN I（SGI）分析，确定适体S2具有最高的亲和力和特异性。基于分子对接探索的结合位点对适体S2进行进一步优化。最终，获得了截短的适体S2-40，其在亲和力和特异性方面均优于S2，解离常数（Kd）分别为6.65±3.07μM（ITC）和42.1±9.34 nM（SGI）。这表明微流控SELEX平台为适体筛选提供了一种更集成、便捷的方法。
