Continuous Separation of DNA Molecules by Size Using Insulator-Based Dielectrophoresis.

Separation of nucleic acids has long served as a central goal of analytical research. Innovations in this field may soon enable the development of rapid, on-site sequencing devices that significantly improve both the availability and accuracy of detailed bioinformatics. However, achieving efficient continuous-flow operation and size-based fractionation of DNA still presents considerable challenges. Current methods have not yet satisfied the need for rapid fractionation of size-heterogeneous nucleic acid samples into specific and narrow size distributions. Dielectrophoretic (DEP) mechanisms integrated in microfluidic devices offer unique advantages for such applications, including short processing times, microscale reaction volumes, and the potential for low cost and portability. To facilitate such developments, we have adapted a microfluidic constriction sorter device to separate a wide range of nucleic acid analytes into distinct microchannel outlets. This work demonstrates selective and tunable deflection of DNA using alternating current (AC) insulator-based dielectrophoresis. We report conditions for size-based DEP sorting of 1.0, 10.2, 19.5, and 48.5 kbp dsDNA analytes, including both plasmid and genomic DNA. Applied potentials range from 200 to 2400 Vpp with frequencies ranging from 50 Hz to 20 kHz. These conditions result in sorting efficiencies up to 92% with a strong dependence on applied potentials and frequencies. In low-frequency AC fields, long DNA molecules form macro-ion clusters. This behavior is associated with an apparent shift from positive to negative DEP sorting behavior. Using a finite element model, we characterize the dynamics of sorting in the microdevice and link differential sorting to differences in dielectrophoretic mobility. We propose the use of a continuous-flow sorting strategy to facilitate future coupling to next generation sequencing approaches.

Concentration of Sindbis virus with optimized gradient insulator-based dielectrophoresis.

Biotechnology, separation science, and clinical research are impacted by microfluidic devices. Separation and manipulation of bioparticles such as DNA, protein and viruses are performed on these platforms. Microfluidic systems provide many attractive features, including small sample size, rapid detection, high sensitivity and short processing time. Dielectrophoresis (DEP) and electrophoresis are especially well suited to microscale bioparticle control and have been demonstrated in many formats. In this work, an optimized gradient insulator-based DEP device was utilized for concentration of Sindbis virus, an animal virus with a diameter of 68 nm. Within only a few seconds, the concentration of Sindbis virus can be increased by two to six times in the channel under easily accessible voltages as low as about 70 V. Compared with traditional diagnostic methods used in virology, DEP-based microfluidics can enable faster isolation, detection and concentration of viruses in a single step within a short time.

Correction: Biophysical separation of Staphylococcus epidermidis strains based on antibiotic resistance.

Correction for 'Biophysical separation of Staphylococcus epidermidis strains based on antibiotic resistance' by Paul V. Jones et al., Analyst, 2015, 140, 5152-5161.

Biophysical separation of Staphylococcus epidermidis strains based on antibiotic resistance.

Electrophoretic and dielectrophoretic approaches to separations can provide unique capabilities. In the past, capillary and microchip-based approaches to electrophoresis have demonstrated extremely high-resolution separations. More recently, dielectrophoretic systems have shown excellent results for the separation of bioparticles. Here we demonstrate resolution of a difficult pair of targets: gentamicin resistant and susceptible strains of Staphylococcus epidermidis. This separation has significant potential implications for healthcare. This establishes a foundation for biophysical separations as a direct diagnostic tool, potentially improving nearly every figure of merit for diagnostics and antibiotic stewardship. The separations are performed on a modified gradient insulator-based dielectrophoresis (g-iDEP) system and demonstrate that the presence of antibiotic resistance enzymes (or secondary effects) produces a sufficient degree of electrophysical difference to allow separation. The differentiating factor is the ratio of electrophoretic to dielectrophoretic mobilities. This factor is 4.6 ± 0.6 × 10(9) V m(-2) for the resistant strain, versus 9.2 ± 0.4 × 10(9) V m(-2) for the susceptible strain. Using g-iDEP separation, this difference produces clear and easily discerned differentiation of the two strains.

Development of the resolution theory for gradient insulator-based dielectrophoresis.

New and important separations capabilities are being enabled by utilizing other electric field-induced forces besides electrophoresis, among these is dielectrophoresis. Recent works have used experimentally simple insulator-based systems that induce field gradients creating dielectrophoretic force in useful formats. Among these, juxtaposing forces can generate gradient-based steady-state separations schemes globally similar to isoelectric focusing. The system of interest is termed gradient insulator-based dielectrophoresis and can create extremely high resolution steady-state separations for particles four nanometers to ten micrometers in diameter, including nearly all important bioparticles (large proteins, protein aggregates, polynucleotides viruses, organelles, cells, bacteria, etc.). A theoretical underpinning is developed here to understand the relationship between experimental parameters and resolution and to identify the best expected resolution possible. According to the results, differences in particles (and bioparticles) as small as one part in 10(4) for diameter (subnanometer resolution for a one micrometer particle), one part in 10(8) for dielectrophoretic parameters (dielectrophoretic mobility, Clausius-Mossotti factor), and one part in 10(5) for electrophoretic mobility can be resolved. These figures of merit are generally better than any competing technique, in some cases by orders of magnitude. This performance is enabled by very strong focusing forces associated with localized gradients.

Differentiation of Escherichia coli serotypes using DC gradient insulator dielectrophoresis.

Bacteria play a significant role in both human health and disease. An estimated 9.4 million cases of foodborne illness occur in the United States each year. As a result, rapid identification and characterization of microorganisms remains an important research objective. Despite limitations, selective culturing retains a central role among a cadre of identification strategies. For the past decade, separations-based approaches to rapid bacterial identification have been under investigation. Gradient insulator dielectrophoresis (g-iDEP) promises benefits in the form of rapid and specific separation of very similar bacteria, including serotypes of a single species. Furthermore, this approach allows simultaneous concentration of analyte, facilitating detection and downstream analysis. Differentiation of three serotypes or strains of Escherichia coli bacteria is demonstrated within a single g-iDEP microchannel, based on their characteristic electrokinetic properties. Whole cells were captured and concentrated using a range of applied potentials, which generated average electric fields between 160 and 470 V/cm. Bacteria remained viable after exposure to these fields, as determined by cellular motility. These results indicate the potential g-iDEP holds in terms of both separatory power and the possibility for diagnostic applications.

Manipulation and capture of Aß amyloid fibrils and monomers by DC insulator gradient dielectrophoresis (DC-iGDEP).

Here we report a novel method for the manipulation and concentration of Aß amyloid fibrils, implicated in Alzheimer's disease, using DC insulating gradient dielectrophoresis (DC-iGDEP). Fibril enrichment was found to be ∼400%. Simulations suggest that capture of the full range of amyloid protein aggregates is possible with optimized device design.

Blood cell capture in a sawtooth dielectrophoretic microchannel.

Biological fluids can be considered to contain information-rich mixtures of biochemicals and particles that enable clinicians to accurately diagnose a wide range of pathologies. Rapid and inexpensive analysis of blood and other bodily fluids is a topic gaining substantial attention in both science and medicine. One line of development involves microfluidic approaches that provide unique advantages over entrenched technologies, including rapid analysis times, microliter sample and reagent volumes, potentially low cost, and practical portability. The present study focuses on the isolation and concentration of human blood cells from small-volume samples of diluted whole blood. Separation of cells from the matrix of whole blood was accomplished using constant potential insulator-based gradient dielectrophoresis in a converging, sawtooth-patterned microchannel. The channel design enabled the isolation and concentration of specific cell types by exploiting variations in their characteristic physical properties. The technique can operate with isotonic buffers, allowing capture of whole cells, and reproducible capture occurred at specific locales within the channel over a global applied voltage range of 200-700 V.

Dielectrophoretic mobility determination in DC insulator-based dielectrophoresis.

Insulator-based dielectrophoresis (iDEP) is a powerful tool for separating and characterizing particles, yet it is limited by a lack of quantitative characterizations. Here, this limitation is addressed by employing a method capable of quantifying the DEP mobility of particles. Using streak-based velocimetry the particle properties are deduced from their motion in a microfluidic channel with a constant electric field gradient. From this approach, the DEP mobility of 1 µm polystyrene particles was found to be -2±0.4 10(-8) cm4 /(V2 s). In the future, such quantitative treatment will allow for the elucidation of unique insights and rational design of devices.