Timothy Olsen

An Integrated Microfluidic SELEX Approach Using Combined Electrokinetic and Hydrodynamic Manipulation.

This article presents a microfluidic approach for the integration of the process of aptamer selection via systematic evolution of ligands by exponential enrichment (SELEX). The approach employs bead-based biochemical reactions in which affinity-selected target-binding oligonucleotides are electrokinetically transferred for amplification, while the amplification product is transferred back for affinity selection via pressure-driven fluid flow. The hybrid approach simplifies the device design and operation procedures by reduced pressure-driven flow control requirements and avoids the potentially deleterious exposure of targets to electric fields prior to and during affinity selection. In addition, bead-based reactions are used to achieve the on-chip coupling of affinity selection and amplification of target-binding oligonucleotides, thereby realizing on-chip loop closure and integration of the entire SELEX process without requiring offline procedures. The microfluidic approach is thus capable of closed-loop, multiround aptamer enrichment as demonstrated by selection of DNA aptamers against the protein immunoglobulin E with high affinity (KD = 12 nM) in a rapid manner (4 rounds in approximately 10 h).

Integrated Microfluidic Isolation of Aptamers Using Electrophoretic Oligonucleotide Manipulation

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.

Integrated Microfluidic Selex Using Free Solution Electrokinetics

Systematic evolution of ligands by exponential enrichment (SELEX) offers a powerful method to isolate affinity oligonucleotides known as aptamers, which can then be used in a wide range of applications from drug delivery to biosensing. However, conventional SELEX methods rely on labor intensive and time consuming benchtop operations. A simplified microfluidic approach is presented which allows integration of the affinity selection and amplification stages of SELEX for the isolation of target-binding oligonucleotides by combining bead-based biochemical reactions with free solution electrokinetic oligonucleotide transfer. Free solution electrokinetics allows coupling of affinity selection and amplification for closed loop oligonucleotide enrichment without the need for offline processes, flow handling components or gel components, while bead based selection and amplification allow efficient manipulation of reagents and reaction products thereby realizing on-chip loop closure and integration of the entire SELEX process. Thus the approach is capable of multi-round enrichment of oligonucleotides using simple transfer processes while maintaining a high level of device integration, as demonstrated by the isolation of an aptamer pool against a protein target (IgA) with significantly higher binding affinity than the starting library in approximately 4 hours of processing time.

An aptameric graphene nanosensor for analyte detection in serum

We present an aptameric graphene field-effect transistor (GFET) nanosensor for sensitive and label-free detection of biomarkers in serum. The graphene nanosensor is serially functionalized with a polyethylene glycol (PEG) nanolayer and an aptamer for specific detection of a target analyte and effective rejection of background molecules in human serum. The analyte binds to the surface-immobilized aptamer, changing the carrier concentration in the graphene. The resulting changes in the conductance of the graphene is measured to determine the analyte concentration. Experimental results show that the device is capable of detecting immunoglobulin E (IgE) in serum in a clinically relevant range of 50 pM to 35 nM.