A comparative study of aptamer isolation by conventional and microfluidic strategies

Abstract

Aptamers are single-stranded oligonucleotide molecules that bind with high affinity and specificity to a wide range of target molecules. The method of systematic evolution of ligands by exponential enrichment (SELEX) plays an essential role in the isolation of aptamers from a randomized oligonucleotide library. To date, significant modifications and improvements of the SELEX process have been achieved, engendering various forms of SELEX from conventional SELEX to microfluidics-based full-chip SELEX. While full-chip SELEX is generally considered advantageous over conventional SELEX, there has not yet been a conclusive comparison between the methods. Herein, we present a comparative study of three SELEX strategies for aptamer isolation, including those using conventional agarose bead-based partitioning, microfluidic affinity selection, and fully integrated microfluidic affinity selection and PCR amplification. Using immunoglobulin E (IgE) as a model target molecule, we compare these strategies in terms of the time and cost for each step of the SELEX process including affinity selection, amplification, and oligonucleotide conditioning. Target-binding oligonucleotides in the enriched pools are sequenced and compared to assess the relative efficacy of the SELEX strategies. We show that the microfluidic strategies are more time- and cost-efficient than conventional SELEX.

Fig. 1

Aptamer selection procedures of (a) conventional SELEX and (b) microfluidic SELEX. Design of (c) the on-chip affinity selection chip and (d) full-chip SELEX chip.

Fig. 2

Amplification characterization of target-binding oligonucleotides. (a) Gel electrophoresis of elution SELEX after PCR (line 1: negative control; line 2: conventional SELEX; line 3: on-chip affinity selection; line 4: full-chip SELEX; and line 5: ladder). Fluorescence images of PCR beads (b) before bead-based PCR and (c) after bead-based PCR in on-chip affinity selection. Fluorescence images of PCR beads (d) before on-chip bead-based PCR and (e) after on-chip bead-based PCR in full-chip SELEX.

Fig. 3

Affinity characterization of the selected aptamer. (a) Determination of the equilibrium dissociation constant for IgE with aptamer IGE1. (b) Secondary structure of aptamer IGE1.

Fig. 4

Affinity characterization of the truncated aptamer. (a) Process of truncating IGE1 into a shorter IGE1-T. (b) Determination of equilibrium dissociation constants of IGE1-T against IgE.

Fig. 5

Specificity of the truncated aptamer IGE1-T against IgE protein.

Fig. 6

Comparison of the three SELEX methods in terms of (a) enrichment, (b) total time, and (c) total cost used for the SELEX process. All results are from round 4 against IgE of each method.