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

Abstract

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.

Dielectrophoresis based continuous-flow nano sorter: fast quality control of gene vaccines

“…2 (a) the associated dielectrophoretic potential is depicted. the analyte had two possible pathways at the ridge; 37 either the analyte passed the nanoslit unhindered or was deflected towards the other channel wall. The dielectrophoretic force is oriented essentially perpendicular to the edge of the ridge, whereas its component tangential to the ridge is very small.…”

“…37,39 Concerning devices for the separation of three or more analytes, three different approaches were considered: (1) Hawkins et al suggested that analytes of different polarizabilities would escape from an arc-shaped ridge at different locations, due to the angle dependent migration. 37,39 Concerning devices for the separation of three or more analytes, three different approaches were considered: (1) Hawkins et al suggested that analytes of different polarizabilities would escape from an arc-shaped ridge at different locations, due to the angle dependent migration.…”

“…37 Here, we extend the principle to multiple separation, i.e. 37 Here, we extend the principle to multiple separation, i.e.…”

Dielectrophoresis for Bioparticle Manipulation

“…Viefhues M et al [112,113] embedded the S-shaped ridge in the microchannel to squeeze the electric field. Due to the disadvantages of the pure DC voltage such as a serious Joule heating effect, AC and DC voltages were used to generate electrokinetic transport and dielectrophoresis effects.…”

Label-Free Biosensing Method for the Detection of a Pancreatic Cancer Biomarker Based on Dielectrophoresis Spectroscopy

“…Kawabata & Washizu exploited the dependence of the DEP force on molecular binding on the surface of functionalized PM to separate PM as a function of the molecular binding for alpha-fetoprotein and DNA strands [24]. DEP was also used to concentrate DNA molecules with [25] and without [26] PM.…”