Integrated Microfluidic Isolation of Aptamers Using Electrophoretic Oligonucleotide Manipulation

Abstract

We present a microfluidic approach to integrated isolation of DNA aptamers via systematic evolution of ligands by exponential enrichment (SELEX). The approach employs a microbead-based protocol for the processes of affinity selection and amplification of target-binding oligonucleotides, and an electrophoretic DNA manipulation scheme for the coupling of these processes, which are required to occur in different buffers. This achieves the full microfluidic integration of SELEX, thereby enabling highly efficient isolation of aptamers in drastically reduced times and with minimized consumption of biological material. The approach as such also offers broad target applicability by allowing selection of aptamers with respect to targets that are either surface-immobilized or solution-borne, potentially allowing aptamers to be developed as readily available affinity reagents for a wide range of targets. We demonstrate the utility of this approach on two different procedures, respectively for isolating aptamers against a surface-immobilized protein (immunoglobulin E) and a solution-phase small molecule (bisboronic acid in the presence of glucose). In both cases aptamer candidates were isolated in three rounds of SELEX within a total process time of approximately 10 hours.

Discovery of tumoricidal DNA oligonucleotides by response-directed in vitro evolution

“…Additional efficient modalities for aptamer selection include capillary electrophoresis [37][38][39] and magnetic sorting 40 . Electrophoresis and magnetic beads have been combined with microfluidics to create integrated systems for aptamer selection [41][42][43] . All these methods have demonstrated that aptamer selection could be significantly sped up and multiplexed.…”

Aptamers against Immunoglobulins: Design, Selection and Bioanalytical Applications

“…However, the dissociation constant of the selected IgE aptamer (83.9 nM) was not as good as in selections made by classical SELEX and CE-SELEX [ 22 ]. A fully microchip integrated version of the electrophoretic selection process by combining the bead-based aptamer selection (bead-based PCR amplification) with gel electrophoresis was improved [ 23 ], resulted in aptamers reaching a K D value of 18 nM in only 3 selection rounds [ 24 ].…”

“…Magnetic separations can be integrated in microfluidics and combined with SELEX (M-SELEX) to ensure an efficient, compact and fast screening of aptamers against the target. These systems were evaluated for the selection of as SIgA [21] and IgE [22][23][24] selective aptamers. Generally, such microfluidic devices comprise two chambers: a selection chamber and an amplification chamber.…”

Microfluidic Technology for Nucleic Acid Aptamer Evolution and Application

“…Subsequently, an integrated microfluidic system was developed by Kim et al that coupled the affinity selection and amplification step by an electrophoretic oligonucleotide manipulation scheme. This allowed for the rapid selection of aptamers against either surface‐immobilized protein (immunoglobulin E) or solution‐phase small molecule (bisboronic acid–glucose mixture) with high affinity …”