Measuring the dielectric properties of herpes simplex virus type 1 virions with dielectrophoresis

An investigation has been performed into the biophysical properties of the enveloped mammalian virus, herpes simplex virus type 1 (HSV-1). The dielectrophoretic behaviour of the virus particles was measured as a function of applied frequency (over the range 100 kHz–20 MHz) and conductivity of the suspending medium (over the range 1–100 mS m⁻¹). The dielectric properties of the virus were determined from the dielectrophoretic data using the smeared-out shell model. The data suggest that the intact particle has a surface conductance of 0.3 nS, an internal and membrane permittivity of 75ε_o and 7.5ε_o, respectively, an internal conductivity of approximately 0.1 S m⁻¹ and a zeta potential of 70 mV.The dielectric properties were measured for intact, fresh virus particles and also for particles following exposure to various modifying agents, such as treatment with enzymes, ionophores and ageing. It is shown that the observed changes in the dielectrophoretic spectrum, and the variations in the dielectric properties of the virus concur with the expected physiological effects of these agents.     

Electrorotation and levitation of cells and colloidal particles

We review dielectrophoretic forces on cells and colloidal particles, emphasizing their use for manipulating and characterizing the electrical properties of suspended particles. Compared with dielectric spectroscopy, these methods offer a measure of independence from electrode artifacts and mixture theory. On the assumption that the particles can be modeled as uniform dielectric objects with effective dielectric properties, a simple theory can be developed for the frequency variation in the field-induced forces. For particles exhibiting counterion polarization, dielectrophoretic forces differ considerably from predictions of this theory at low frequencies, apparently because of double layer phenomena.     

Dielectrophoretic force: A comparison of theory and experiment

Because of the increasing use of dielectrophoresis in the dielectric characterization and sorting of living cells or their parts, it has become important to establish carefully the theoretical backgrounds for this effect. A comparison with experiment is made of the several versions of the theory for the dielectrophoretic force exerted by nonuniform electric fields upon a neutral object. The three fundamental approaches: the Maxwell-Strattonstress tensor, the effective dipole moment, and the ‘Helmholtz’ energy approach are presented along with the general solution given earlier by Pohl and Crane. These are found to agree closely with experiment in predicting the dielectrophoretic force upon various rods hung in specially shaped (isomotive) field distributions. On the other hand, an alternative formulation based upon a debatable assignment of fields local to the dipoles gave a good fit to the experimental data only for materials of very low permittivity, and fitted poorly in the case of highly polarizable materials. An improved derivation of the theory for stable dielectrophoretic levitation is also presented. This phenomenon is of particular interest in that it is based upon an apparent violation of the Earnshaw's theorem, and is useful in the study of the dielectric properties of individual living cells.     

A Nomograph for Calculating the Optimal Frequency for Dielectrophoresis and the Characteristic Frequency of the β-Dispersion of Cell Membrane Vesicles

The first stage of the two-stage cell electrofusion technique involves the dielectrophoretic apposition, in an AC field, of protoplasts suspended in a medium of relatively low specific conductivity. A frequency at which the maximum dielectrophoretic force is exerted is given by the characteristic frequency for the dielectric relaxation by a Maxwell-Wagner type of mechanism. We provide a nomograph for the rapid calculation of this frequency.     

Membrane dielectric changes indicate induced apoptosis in HL-60 cells more sensitively than surface phosphatidylserine expression or DNA fragmentation

The specific membrane capacitance and conductivity of mammalian cells, which reflect their surface morphological complexities and membrane barrier functions, respectively, have been shown to respond to cell physiologic and pathologic changes. Here, the effects of induced apoptosis on these membrane properties of cultured human promyelocytic HL-60 cells are reported. Changes in membrane capacitance and conductivity were deduced from measurements of cellular dielectrophoretic crossover frequencies following treatment with genistein (GEN). The apparent specific cell membrane capacitance of HL-60 cells fell from an initial value of 17.6+/-0.9 to 9.1+/-0.5 mF/m(2) 4 h after treatment. Changes began within minutes of treatment and preceded both the externalization of phosphatidylserine (PS), as gauged by the Annexin V assay, and the appearance of a sub-G1 cell subpopulation, as determined through ethidium bromide staining of DNA. Treatment by the broad spectrum caspase inhibitor N-benzyloxycarbony-Val-Ala-Asp(O-methyl)-fluoromethyketone (zVAD-fmk) did not prevent these early cell membrane dielectric responses, suggesting that the caspase system was not involved. Although membrane conductivity did not alter during the first 4 h of GEN treatment, it rose significantly and progressively thereafter. Finally, as the barrier function failed and the cells became necrotic, it increased by many orders of magnitude. The effective membrane capacitance and conductivity findings serve to focus attention on the membrane as a site for early participation in apoptosis. In conjunction with our prior reports of the use of dielectric methods for cell manipulation and separation, these results demonstrate that dielectrophoretic technologies should be applicable to the rapid detection, separation, and quantification of normal, apoptotic, and necrotic cells from cell mixtures.     

Membrane dielectric changes indicate induced apoptosis in HL-60 cells more sensitively than surface phosphatidylserine expression or DNA fragmentation

The specific membrane capacitance and conductivity of mammalian cells, which reflect their surface morphological complexities and membrane barrier functions, respectively, have been shown to respond to cell physiologic and pathologic changes. Here, the effects of induced apoptosis on these membrane properties of cultured human promyelocytic HL-60 cells are reported. Changes in membrane capacitance and conductivity were deduced from measurements of cellular dielectrophoretic crossover frequencies following treatment with genistein (GEN). The apparent specific cell membrane capacitance of HL-60 cells fell from an initial value of 17.6±0.9 to 9.1±0.5 mF/m² 4 h after treatment. Changes began within minutes of treatment and preceded both the externalization of phosphatidylserine (PS), as gauged by the Annexin V assay, and the appearance of a sub-G1 cell subpopulation, as determined through ethidium bromide staining of DNA. Treatment by the broad spectrum caspase inhibitor N-benzyloxycarbony-Val-Ala-Asp(O-methyl)-fluoromethyketone (zVAD-fmk) did not prevent these early cell membrane dielectric responses, suggesting that the caspase system was not involved. Although membrane conductivity did not alter during the first 4 h of GEN treatment, it rose significantly and progressively thereafter. Finally, as the barrier function failed and the cells became necrotic, it increased by many orders of magnitude. The effective membrane capacitance and conductivity findings serve to focus attention on the membrane as a site for early participation in apoptosis. In conjunction with our prior reports of the use of dielectric methods for cell manipulation and separation, these results demonstrate that dielectrophoretic technologies should be applicable to the rapid detection, separation, and quantification of normal, apoptotic, and necrotic cells from cell mixtures.     

Separation of submicron bioparticles by dielectrophoresis.

Submicron particles such as latex spheres and viruses can be manipulated and characterized using dielectrophoresis. By the use of appropriate microelectrode arrays, particles can be trapped or moved between regions of high or low electric fields. The magnitude and direction of the dielectrophoretic force on the particle depends on its dielectric properties, so that a heterogeneous mixture of particles can be separated to produce a more homogeneous population. In this paper the controlled separation of submicron bioparticles is demonstrated. With electrode arrays fabricated using direct write electron beam lithography, it is shown that different types of submicron latex spheres can be spatially separated. The separation occurs as a result of differences in magnitude and/or direction of the dielectrophoretic force on different populations of particles. These differences arise mainly because the surface properties of submicron particles dominate their dielectrophoretic behavior. It is also demonstrated that tobacco mosaic virus and herpes simplex virus can be manipulated and spatially separated in a microelectrode array.     

Dielectric properties of human leukocyte subpopulations determined by electrorotation as a cell separation criterion.

The separation and purification of human blood cell subpopulations is an essential step in many biomedical applications. New dielectrophoretic fractionation methods have great potential for cell discrimination and manipulation, both for microscale diagnostic applications and for much larger scale clinical problems. To discover whether human leukocyte subpopulations might be separable by such methods, the dielectric characteristics of the four main leukocyte subpopulations, namely, B- and T-lymphocytes, monocytes, and granulocytes, were measured by electrorotation over the frequency range 1 kHz to 120 MHz. The subpopulations were derived from human peripheral blood by magnetically activated cell sorting (MACS) and sheep erythrocyte rosetting methods, and the quality of cell fractions was checked by flow cytometry. Mean specific membrane capacitance values were calculated from the electrorotation data as 10.5 (+/- 3.1), 12.6 (+/- 3.5), 15.3 (+/- 4.3), and 11.0 (+/- 3.2) mF/m2 for T- and B-lymphocytes, monocytes, and granulocytes, respectively, according to a single-shell dielectric model. In agreement with earlier findings, these values correlated with the richness of the surface morphologies of the different cell types, as revealed by scanning electron microscopy (SEM). The data reveal that dielectrophoretic cell sorters should have the ability to discriminate between, and to separate, leukocyte subpopulations under appropriate conditions.     

The ac field patterns about living cells

The presence of the small ac electric fields produced by living cells is shown by the gentle dielectrophoretic force on tiny dielectric particles. The beauty of the field patterns is made evident as the cell and particles settle in a hanging drop. Patterns characteristic of repulsion and of attraction, as well as of a dipolar and quadripolar nature, are observable.     

The ac field patterns about living cells

The presence of the small ac electric fields produced by living cells is shown by the gentle dielectrophoretic force on tiny dielectric particles. The beauty of the field patterns is made evident as the cell and particles settle in a hanging drop. Patterns characteristic of repulsion and of attraction, as well as of a dipolar and quadripolar nature, are observable.     

Determination of electric parameters of cell membranes by a dielectrophoresis method.

Marszalek, P., J. J. Zielinsky, and M. Fikus (1989. Bioelectrochem. Bioenerg. 22:289-298) have described a novel design for measuring the complete dielectrophoretic spectrum of a single cell. From the analysis of the dielectrophoretic spectrum, the membrane conductivity, sigma membr, and the membrane dielectric permittivity, epsilon membr, of the cell may be determined according to the theory of dielectrophoresis described by Sauer, F. A. (1985. Interactions between Electromagnetic Field and Cells. A. Chiabrera, C. Nicolini, and H.P. Schwan, editors. Plenum Publishing Corp., New York. 181-202). At Fo, the net force experienced by a single shell sphere in a nonuniform periodic field is zero, and the sphere ceases to move in the field. In other words, at Fo, the effective polarizability, chi eff, of the sphere (the polarizability of sphere minus the polarizability of the medium) is equal to zero. For biological cells in high conductivity medium, e.g., the isotonic saline, sigma membr falls below 2 x 10(-6) S m-1, where Fo becomes insensitive to sigma membr, and the method becomes impractical. In a low conductivity medium, 0.3 M sucrose, sigma membr of cells is generally higher and the method may be applied. Assuming a membrane thickness of 9 nm, epsilon membr of Neurospora crassa slime cells was determined to be in the range of 8.3-9.4 epsilon o, and of myeloma Tib9 to be 9.4 epsilon o, epsilon o being the dielectric permittivity of the free space. The values for the slime cells were compared with values obtained by the dielectric spectroscopy method which measures average values for cells in suspension.     

Dielectrophoretic study of human erythrocytes

This paper is concerned with the dielectrophoretic study of human erythrocytes under cylindrical field geometry. The influence of physical variables such as the frequency and voltage of the applied electric field, conductivity of the medium in which the cells are suspended, cell concentration and exposure time of the cell to the non-uniform electric field on dielectrophoretic collection rate (DCR) is determined in a systematic manner. It is interesting to note from the DCR spectrum of human erythrocytes that the DCR is minimum at one frequency, maximum at another and there is practically no yield over a certain frequency range. This may be attributed to the variation of complex dielectric constant of the particle and medium over that frequency range. From the DCR spectrum of different groups, it is clear that DCR behaviour is different in the frequency range from 0.3–1.5 MHz, under similar conditions of temperature, conductivity and concentration of erythrocyte suspension and strength of applied AC field. The response of DCR with voltage of the applied field, concentration of cell suspension and square root of elapsed time of the cells confirms the theory of dielectrophoresis.     

Trapping, deformation, and rotation of giant unilamellar vesicles in octode dielectrophoretic field cages

The behavior of freestanding lipid bilayer membranes under the influence of dielectric force potentials was studied by trapping, holding, and rotating individual giant unilamellar vesicles (GUVs) inside dielectrophoretic microfield cages. Using laser scanning confocal microscopy and three-dimensional image reconstructions of GUVs labeled with fluorescent membrane probes, field strength and frequency-dependent vesicle deformations were observed which are explained by calculations of the dielectric force potentials inside the cage. Dynamical membrane properties under the influence of the field cage were studied by fluorescence correlation spectroscopy, circumventing potential artifacts associated with measurements involving GUV immobilization on support surfaces. Lipid transport could be accelerated markedly by the applied fields, aided by hydrodynamic fluid streaming which was also studied by fluorescence correlation spectroscopy.     

Dielectrophoresis as a tool to characterize and differentiate isogenic mutants of Escherichia coli

In this study we report on an experimental method based on dielectrophoretic analysis to identify changes in four Escherichia coli isogenic strains that differed exclusively in one mutant allele. The dielectrophoretic properties of wild-type cells were compared to those of hns, hha, and hha hns mutant derivatives. The hns and hha genes code respectively for the global regulators Hha and H-NS. The Hha and H-NS proteins modulate gene expression in Escherichia coli and other Gram negative bacteria. Mutations in either hha or hns genes result in a pleiotropic phenotype. A two-shell prolate ellipsoidal model has been used to fit the experimental data, obtained from dielectrophoresis measurements, and to study the differences in the dielectric properties of the bacterial strains. The experimental results show that the mutant genotype can be predicted from the dielectrophoretic analysis of the corresponding cultures, opening the way to the development of microdevices for specific identification. Therefore, this study shows that dielectrophoresis can be a valuable tool to study bacterial populations which, although apparently homogeneous, may present phenotypic variability.     

A comparative study of cell death using electrical capacitance measurements and dielectrophoresis

The changes in the dielectric properties of cells that occur during their exposure to various lethal environmental stresses were measured using both dielectric spectroscopy and dielectrophoresis. It is shown that the dielectric properties of both dying and dead yeast cells were strongly dependent on the method used to induce cell death. Methods which directly affected the membrane permeability, and consequently the membrane conductivity and internal conductivity, resulted in large changes in the suspension capacitance and dielectrophoretic behaviour, whilst methods which affected the cell interior but had little effect on the cell membrane resulted in few or no changes in the dielectric properties of the cells. The findings indicate that, depending on the method by which cell death is induced, dielectric spectroscopy may not always be able to observe differences between viable and non-viable cells, and that dielectrophoresis will not always be able to separate viable from non-viable cells.     

Dielectric characterization of bacterial cells using dielectrophoresis

Measurements of dielectrophoretic collection spectra of Escherichia coli and Staphylococcus aureus suspensions are used for obtaining dielectric characteristics of both types of bacteria. The experiments are interpreted using a numerical method that models the cells as compartmented spherical or rod-like particles. We show the usefulness of this simple method to extract significant information about the electrical properties of Gram-negative and -positive bacteria.     

Citric Acid Fermentation

Abstract

Dielectrophoresis is the motion of particles caused by electrical polarization effects in inhomogeneous (nonuniform) electric fields. Unlike electrophoresis, the particles do not require a net electrical charge for motion to occur and AC rather than DC fields are employed to exploit the dielectric properties of the particles. Factors controlling the effective dielectric properties of cells and microorganisms include electrical double layers associated with surface charges, the conductivity and permittivity of their membranes and any cell walls, and their morphologies and structural architectures. In recent years, several laboratories have developed separation and manipulation techniques for cells and microorganisms based on dielectrophoresis, using both static and traveling AC fields. In this article, the basic physical factors influencing the dielectrophoretic behavior of particles are outlined, and ways in which these can be employed to achieve selective separation of cells and microorganisms are described.     

Development of dielectrophoresis separator with an insulating porous membrane using DC‐Offset AC Electric Fields

Our previous studies revealed that the dielectrophoresis method is effective for separating cells having different dielectric properties. The purpose of this study was to evaluate the separation characteristics of two kinds of cells by direct current (DC) voltage offset/alternating current (AC) voltage using an insulating porous membrane dielectrophoretic separator. The separation device gives dielectrophoretic (DEP) force and electrophoretic (EP) force to dispersed particles by applying the DC‐offset AC voltage. This device separates cells of different DEP properties by adopting a structure in which only the parallel plate electrodes and the insulating porous membrane are disposed in the flow path through which the cell‐suspension flows. The difference in the retention ratios of electrically homogeneous 4.5 μm or 20.0 μm diameter standard particles was a maximum of 82 points. Furthermore, the influences of the AC voltage or offset voltage on the retention ratios of mouse hybridoma 3‐2H3 cells and horse red blood cells (HRBC) were investigated. The difference in the retention ratio of the two kinds of cells was a maximum of 56 points. The separation efficiency of this device is expected to be improved by changing the device shape, number of pores, and pore placement. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1292–1300, 2016     

Electrodeless dielectrophoresis of single- and double-stranded DNA

Dielectrophoretic trapping of molecules is typically carried out using metal electrodes to provide high field gradients. In this paper we demonstrate dielectrophoretic trapping using insulating constrictions at far lower frequencies than are feasible with metallic trapping structures because of water electrolysis. We demonstrate that electrodeless dielectrophoresis (EDEP) can be used for concentration and patterning of both single-strand and double-strand DNA. A possible mechanism for DNA polarization in ionic solution is discussed based on the frequency, viscosity, and field dependence of the observed trapping force.     

Dielectrophoretic separation of monocytes from cancer cells in a microfluidic chip using electrode pitch optimization

This study proposes a microfluidic device capable of separating monocytes from a type of cancer cell that is called T-cell acute lymphoblastic leukemia (RPMI-8402) in a continuous flow using negative and positive dielectrophoretic forces. The use of both the hydrodynamic and dielectrophoretic forces allows the separation of RPMI-8402 from monocytes based on differences in their intrinsic electrical properties and sizes. The specific crossover frequencies of monocytes and RPMI-8402 cells have been obtained experimentally. The optimum ranges of electrode pitch-to-channel height ratio at the cross sections with different electrode widths have been generally calculated by numerical simulations of the gradients of the electric field intensities and calculation their effective values (root-mean-square). In the device, the cell sorting has been conducted empirically, and then, the separation performance has been evaluated by analyzing the images before and after dielectrophoretic forces applied to the cells. In this work, the design of a chip with 77 μm gold–titanium electrode pitch was investigated to achieve high purity of monocytes of 95.2%. The proposed device can be used with relatively low applied voltages, as low as 16.5 V (peak to peak). Thus, the design can be used in biomedical diagnosis and chemical analysis applications as a lab-on-chip platform. Also, it can be used for the separation of biological cells such as bacteria, RNA, DNA, and blood cells.