Fast and continuous-flow separation of DNA-complexes and topological DNA variants in microfluidic chip format

The efficient detection, separation and purification of topological and (protein-)complexed DNA variants is mandatory for many state-of-the-art molecular medicine technologies, like medical diagnostics, gene- and cancer-therapy as well as plasmid vaccination. Here, we present the proof-of-concept of a novel micro-nanofluidic device for a fast and efficient, continuous-flow, and virtually label-free detection/purification protocol that goes beyond the standard methods of electrophoretic mobility shift assays, capillary electrophoresis and affinity chromatography. Based on dielectrophoretic trapping, analyte mixtures of small linear DNA-fragments (2.868 kbp and 6.0 kbp), topological DNA variants like plasmids (6.766 kbp) and minicircle-DNA (2.257 kbp), or cytostatic- and protein-DNA complexes were separated in the vicinity of a channel-spanning bowed ridge (creating a nanoslit). One analyte is continuously deflected due to dielectrophoretic trapping at the ridge whereas other species pass the nanoslit unhindered, resulting in two molecule specific pathways with baseline separated resolution. This offers one-step real-time separation of low analyte volumes on a one-minute timescale at low-costs. The underlying dielectrophoretic mechanism was quantified by determining the electrical polarizabilities of the molecules. Additionally, we compared the continuous-flow detection of DNA-complexes with well-established electrophoretic mobility shift assays. Future analytical and preparative applications, such as for plasmid pharmaceuticals as well as continuous sample harvesting in parallel microchip format, are discussed.