High-efficiency selection of aptamers for bovine lactoferrin by capillary electrophoresis and its aptasensor application in milk powder.

Capillary electrophoresis-based systematic evolution of ligands by exponential enrichment (CE-SELEX) is a high-efficient technique for aptamers selection, and has been evolved into many modes. In this study, we obtained the aptamer against bovine lactoferrin (BLF) with high affinity (dissociation constant, Kd = 20.74 ± 6.89 nM) and good specificity (>1000 folds) using single step CE-SELEX (ssCE-SELEX) mode. In the selection process, ssCE demonstrated high-efficiency selection with bulk Kd reaching at 0.19 ± 0.04 μM by only two rounds, as compared to capillary zone electrophoresis (CZE) mode with Kd of 0.39 ± 0.03 μM. Next-generation sequencing (NGS) was performed by two methods of high output (Hiseq) and medium output (Miseq) with different sequencing depths, and their same results of high-frequency sequences confirmed the reliability of the obtained sequences. Through affinity analysis, the primer region and single base mutation (SBM) were observed to affect the sequence structure and to result in affinity change. Besides, molecular dynamics (MD) simulation was performed to validate the binding affinity of the candidates with BLF by analyzing binding sites, interaction forces, and binding free energy. Moreover, BLF detection in milk powder matrices was completed successfully with the optimized CE-aptasensor. The signal response was in a good linear relationship (R2 = 0.9930) with 4-128 nM of BLF and the detection limit was 1 nM. The obtained results of BLF in four milk powder samples were in an acceptable agreement with the labeled concentrations. This study presented a completed CE based process including aptamers selection, affinity characterization, and detection application, which also validated the high-efficiency selection of ssCE-SELEX mode.