Online reaction based single-step capillary electrophoresis-systematic evolution of ligands by exponential enrichment for ssDNA aptamers selection

Highlights: • The ssCE-SELEX completes six procedures of mixture, incubation, reaction, separation, detection and collection in one run. • One CE run consumes about 60 nL of ssDNA and protein sample, and completes within 15 min, less than those of other methods. • The affinity of the obtained aptamer with K_d of 52.5 ± 24.7 nM is validated separately by ITC, AuNPs, and MD simulation. -- Abstract: Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) has proven to be an effective technique for aptamers selection. In this study, we present an online reaction based convenient single-step CE-SELEX (ssCE-SELEX) mode with human thrombin (H-Thr) as a model target. The selection progress was monitored through bulk K_d analysis, which showed more than a 1000-fold improvement over the initial library after two rounds of selection. Three selected candidate sequences presented high binding affinities against H-Thr with nanomolar (nM) K_d determined by nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM, 56.4–177.1 nM) and CE based non-linear fitting (CE-NLF, 98.2–199.7 nM). They also exhibited high specificities towards H-Thr compared with bovine thrombin, IgG, lysozyme, and lactoferrin. Meanwhile, the K_d results by isothermal titration calorimetry (ITC) confirmed the effective CE in measuring the aptamer affinity. In addition, three candidates were applied as aptasensors in the AuNPs based colorimetric assay, which showed visible color change and good linear relationships (R² > 0.93) with H-Thr concentration. Furthermore, molecular dynamics (MD) simulation was performed to validate the binding of the three candidates with H-Thr by binding sites and binding free energy. The ssCE-SELEX method avoids off-line incubation, saves time and sample, and may provide a universal and convenient method for aptamers selection.