Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu, Taiwan.
Institute of Clinical Medicine, National Cheng Kung University Hospital, National Cheng Kung University, Tainan, Taiwan; Division of General Surgery, Department of Surgery, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Anal Chim Acta. 2023 Sep 15;1274:341531. doi: 10.1016/j.aca.2023.341531. Epub 2023 Jun 21.
Variants of the severe acute respiratory syndrome coronavirus (SARS-CoV-2) have evolved such that it may be challenging for diagnosis and clinical treatment of the pandemic coronavirus disease-19 (COVID-19). Compared with developed SARS-CoV-2 diagnostic tools recently, aptamers may exhibit some advantages, including high specificity/affinity, longer shelf life (vs. antibodies), and could be easily prepared. Herein an integrated microfluidic system was developed to automatically carry out one novel screening process based on the systematic evolution of ligands by exponential enrichment (SELEX) for screening aptamers specific with SARS-CoV-2. The new screening process started with five rounds of positive selection (with the S1 protein of SARS-CoV-2). In addition, including non-target viruses (influenza A and B), human respiratory tract-related cancer cells (adenocarcinoma human alveolar basal epithelial cells and dysplastic oral keratinocytes), and upper respiratory tract-related infectious bacteria (including methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae), and human saliva were involved to increase the specificity of the screened aptamer during the negative selection. Totally, all 10 rounds could be completed within 20 h. The dissociation constant of the selected aptamer was determined to be 63.0 nM with S1 protein. Limits of detection for Wuhan and Omicron clinical strains were found to be satisfactory for clinical applications (i.e. 4.80 × 10 and 1.95 × 10 copies/mL, respectively). Moreover, the developed aptamer was verified to be capable of capturing inactivated SARS-CoV-2 viruses, eight SARS-CoV-2 pseudo-viruses, and clinical isolates of SARS-CoV-2 viruses. For high-variable emerging viruses, this developed integrated microfluidic system can be used to rapidly select highly-specific aptamers based on the novel SELEX methods to deal with infectious diseases in the future.
严重急性呼吸综合征冠状病毒 (SARS-CoV-2) 的变体已经进化，这可能使得对大流行冠状病毒病-19 (COVID-19) 的诊断和临床治疗具有挑战性。与最近开发的 SARS-CoV-2 诊断工具相比，适体可能具有一些优势，包括高特异性/亲和力、更长的保质期（与抗体相比），并且易于制备。在此，开发了一种集成微流控系统，用于自动进行基于指数富集的配体系统进化 (SELEX) 的新型 SARS-CoV-2 特异性适体筛选过程。新的筛选过程从五轮正选择（使用 SARS-CoV-2 的 S1 蛋白）开始。此外，还包括非靶病毒（流感 A 和 B）、人类呼吸道相关癌细胞（腺癌人肺泡基底上皮细胞和发育不良的口腔角质细胞）以及上呼吸道相关传染性细菌（包括耐甲氧西林金黄色葡萄球菌、铜绿假单胞菌、鲍曼不动杆菌和肺炎克雷伯菌），以及人类唾液，以在负选择过程中提高筛选适体的特异性。总共，所有 10 轮可以在 20 小时内完成。选择的适体与 S1 蛋白的解离常数为 63.0 nM。发现武汉和奥密克戎临床株的检测限可满足临床应用的要求（分别为 4.80×10 和 1.95×10 拷贝/mL）。此外，所开发的适体被证明能够捕获灭活的 SARS-CoV-2 病毒、8 种 SARS-CoV-2 假病毒和 SARS-CoV-2 临床分离株。对于高度变异的新兴病毒，这种开发的集成微流控系统可以用于根据新型 SELEX 方法快速选择高度特异性的适体，以应对未来的传染病。
