Chloroplast dielectrophoresis

Dielectrophoretic coefficients of chloroplasts, untreated and treated with cationized ferritin, have been measured in axisymmetric ac electric fields at different frequencies. The treated chloroplasts have surface charge density 2.4 times smaller than the untreated ones.The dielectrophoretic coefficients are in the range 10^-25 F · m² to 7x10^-25 F · m² for frequencies from 6,000 Hz to 1 MHz. Dielectrophoretic effects have not been observed for frequencies from 1 to 6,000 Hz and from 1 MHz and 10 MHz. The surface charge decrease leads to an increase of 2–3 times in the dielectrophoretic coefficients and slight shift of the dielectrophoretic mobility of lower frequencies.These results may be qualitatively explained on the basis of the existing theories for cell and vesicle dielectrophoresis.Abbreviations DCMU 3-(3,4-Dichlorophenyl)-1,1-Dymethylurea - EDTA Ethylenediaminetetraacetic acid - HEPES (N-2 Hydroxyethylpiperozine-N-2-ethanesulphonic acid) - MES (2[N-morpholino]ethane sulfonic acid) - TRICINE (N-tris[Hydroxymethyl]methyl glycine) - DMPA (N,N-dimethyl-1,3-propanediamine)     

The dielectrophoresis of blood platelets as affected by hemophilic traits

We find that there are moderate differences in the electrical polarizabilities of normal and various hemophilic canine blood platelets. The technique of dielectrophoresis, using the effect of nonuniform fields on neutral bodies, was used to perform rapid assays of the platelets. At present the dielectrophoretic test can only distinguish reliably between relatively large groups of animals on a statistical basis. The present technique shows a unique ability however, to distinguish even between normal canine platelets and the transmitter female canine platelets. In studies of the effects of various chemical agents upon the dielectrophoresis of platelets, inhibitors such as NaF, sodium iodacetate, NaCN, and NaN₃ had marked effects at low concentration. Ions such as Na⁺ K⁺, Mg⁺⁺, and La⁺⁺⁺, as well as NO₃ ⁻, SO₄ ⁼, and mellitic ion had lesser effects. In some cases the presence of trace quantities of the chemical agent “stabilizes” the cellular dielectrophoretic response, enabling the platelet to continue to be attracted by the nonuniform field for longer than usual. The CN⁻ and F⁻ ions appear to do this. This may have useful application. From the shape of the frequency spectrum of the dielectrophoretic response we suggest that the peaks at about 0.1 to 10 MHz imply a Maxwell-Wagner type of response, typical of an interface between bulk regions of differing conductivity, as at the cell boundary. From a lack of low frequency response, we suggest that the platelet interface with the surrounding aqueous medium must be singularly free of ionic double layers — or at least that the ionic double layers present must be of unusually low charge density. The technique of dielectrophoresis has been used in comparative study of canine blood platelets, from (1) normal dogs, (2) female dogs that transmit Factor VIII deficiency to their offspring, (3) male dogs with Factor VIII deficiency, and (4) female dogs with Factor VIII deficiency. The study showed that differences exist between the 4 groups of dogs in the average dielectrophoretic responses at 1 MHz. The effect of several chemical agents, i.e., NaCN and NaF on normal canine platelets was to effectively stabilize the platelets against deteriorationin vitro.     

Theoretical and experimental study of the membrane pore effect on the amplitude-frequency dependence of polarizability of a cell

The amplitude-frequency dependence of the polarizability of erythrocytes, yeast cells, and latex particles in the range of 1–10⁶ Hz was studied by the method of dielectrophoresis (DEP). Positive DEP of erythrocytes and yeast cells in a frequency range of 60–100 Hz was revealed. The positive DEP of cells in the given range is theoretically explained by appearance of a great number of transverse pores through the membrane and wall of the cell.     

Levitation, holding, and rotation of cells within traps made by high-frequency fields.

Biological cells and other particles can be electrically manipulated by means of negative dielectrophoresis within microchambers whose electrode geometry is of the order of the cell size. Very-high-frequency fields (50 MHz and above) and media of increased relative permittivity are especially suitable for the purpose, as shown by experimental data on levitation and rotation. It appears to be possible to move and rotate cells or particles at will using this technology.     

Effects of la on surface charges, dielectrophoresis, and electrofusion of barley protoplasts

When dielectrophoresis and electrofusion of barley (Hordeum vulgare var Moor) leaf protoplasts were assayed in the presence of 0.1 to 1 millimolar lanthanum ion (La³⁺) in the basal medium (0.7 molar mannitol, 1 millimolar piperazine-N, N-bis[2-ethanesulfonic acid]-Na [pH 6.7], 0.1 millimolar CaCl₂), dielectrophoresis and induction of electrofusion were strongly inhibited. The latter remained inhibited and the former recovered by about 60% after washing the La³⁺ -treated protoplasts without EDTA. These inhibitions were almost completely abolished by washing the La³⁺ -treated protoplasts with 1 millimolar EDTA. Inductively coupled plasma atomic emission spectroscopic analysis revealed that protoplasts retained a considerable amount of La³⁺ after washing without EDTA and released most of the bound La³⁺ by washing with 1 millimolar EDTA. This tightly bound La³⁺ seemed responsible for the inhibition of electrofusion and dielectrophoresis that was observed in the La³⁺ -treated protoplasts after washing. ζ-potentials of protoplasts were -39.0±3.2 millivolts, -16.7 ± 2.6 millivolts, and virtually zero in media containing 0, 0.1, and 0.3 millimolar La³⁺ (I = 7.2 millimolar), respectively, and had a positive value (+ 14.2 ± 2.2 millivolts) in the presence of 1 millimolar La³⁺. These effects of La³⁺ on ζ-potentials were easily abolished by washing without EDTA. This indicates that charged species located at the surface of plasma membrane of protoplasts cannot account for the sites at which La³⁺ exerts its inhibition of dielectrophoresis and electrofusion. In contrast, the promotion of spherical fusion and the reduction of broken fusion products observed in the presence of La³⁺ were almost completely abolished by washing without EDTA. Our results also indicate that the initial induction and development of electrofusion can be studied independently.     

Dielectric studies on self-associating nucleosides and bases in aqueous solution

Measurements have been made of the dielectric properties of aqueous solutions in which aggregates are formed by stacking. The nucleosides cytidine, uridine and thymidine, and the bases purine, pyrimidine and 6-methylamino-9-methyl-purine (N⁶N⁹-dimethyladenine) were investigated at around 1 MHz, where the static increments can be determined, and for cytidine, dimethyladenine, uridine and pyrimidine measurements were also made in the 100–2000 MHz range where the main relaxation of the solute dipoles is found. Whereas cytidine and purine show a positive static dielectric increment increasing linearly with concentration, dimethyladenine, uridine, thymidine and pyrimidine show a similar negative effect. Also, within the experimental accuracy, single retaxation times are found for the solute dispersions investigated.It is suggested that these relaxations correspond to the effects of free rotation of individual polar molecules in the plane of stacking. This phenomenon would also account for the linear variation of the dielectric increments with concentration. These increments are thought to be positive or negative due to the varying balance in the solutions between the loss of polarization due to displaced and “bound” water and the corresponding gain due to the polarity of the solute molecules.     

Introducing phase analysis light scattering for dielectric characterization: measurement of traveling-wave pumping

Phase analysis light scattering (PALS) was applied to characterize a high-frequency traveling-wave (TW) micropump. Field strength and frequency characteristics were measured for aqueous solutions up to 40 MHz and conductivities of 16 mS/m. The TW field was generated by an ultramicroelectrode array of intercastellated electrodes, which were driven by square-topped signals. Pumping exhibited one major relaxation peak, which strongly increased for conductivities above 4 mS/m. The conductivity dependence of the peak frequency showed an unexpected nonlinear behavior. Around 20 MHz an additional peak caused by electronic resonance was found. Additional coils or capacitors shifted the resonance peak and allowed us to determine the electronic properties of the array. Analysis of distortions in the pump spectra caused by the harmonic content of the driving signals showed that the pump direction is determined by the traveling direction of the field. For measurement of AC-field-induced particle translations, the advantage of PALS over the commonly used microscopic analysis is that it offers an objective method for statistically significant, computerized registration of extremely slow motions. Thus, for dielectric characterization, low field strengths can be used, which is advantageous not only for analyzing liquid pumping, but also for measuring particle translations induced by dielectrophoresis or TW dielectrophoresis.     

Theoretical and experimental study of the effect of membrane pores on spectra of amplitudes of polarization of a cell

Spectra of amplitudes of polarization of erythrocytes, yeast cells, and latex particles in the range of 1-10 Hz were investigated by the method of dielectrophoresis. Positive dielectrophoresis of erythrocytes and yeast cells the frequency range of 60 - 100 Hz was revealed. The theoretically positive dielectrophoresis was evidenced by the occurrence of channels across the cell membrane and bacterial cell wall.     

Tissue engineering with electric fields: immobilization of mammalian cells in multilayer aggregates using dielectrophoresis

Positive dielectrophoresis can be used to create aggregates of animal cells with 3D architectures. It is shown that the cells, when pulled together into an aggregate by positive dielectrophoresis in a low-conductivity iso-osmotic solution, adhere to each other. The adherence of the cells to each other is non-specific and increases in time, and after 10-15 min becomes strong enough to immobilize the cells in the aggregate, enabling the ac electric field to be released, and the iso-osmotic buffer to be replaced by growth or other media. Cell viability is maintained. The new method of immobilization significantly simplifies the construction of aggregates of animal cells by dielectrophoresis, and increases the utility of dielectrophoresis in tissue engineering and related areas.     

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.     

Novel electrode structures for large scale dielectrophoretic separations based on textile technology

The use of dielectrophoresis (DEP) to date has mainly been limited to processing small volumes due to difficulties in the fabrication of microelectrodes over large surface areas. To overcome this problem a novel approach to the construction of micro-electrode arrays has been developed based on weaving. A plain weave cloth was made from 100 microm diameter stainless steel wires and 75 decitex polyester yarns. The stainless steel wires formed the weft, and were kept parallel and apart by a warp of flexible polyester yarns, with a gap of around 150 microm between the metal wires. The metal wires were alternately connected to earth and signal of an AC power source, and it was shown that it was possible to collect yeast cells suspended in deionised water at the metal wire surfaces by dielectrophoresis. The polyester yarn was also found to distort the electric field, creating further areas of electric field non-uniformity around the polyester yarns, further enhancing the capability of the system to attract cells. A 14 ml separation chamber was built from the cloth by alternately sandwiching perspex slabs and cloth together. The DEP chamber was able to effectively collect life yeast from a flow of suspended cells through the cloth using an applied field of 1 MHz at flow rates up to 5 ml min-1. However, some loss occurred due to sedimentation. Also, the chamber was able to separate dead and live yeast cells at 30 Vpk-pk, 2 MHz, with some cell loss due to sedimentation.     

Manipulation of microparticles for construction of array patterns by negative dielectrophoresis using multilayered array and grid electrodes

In this study, a useful method was developed to fabricate array patterns of microparticles not on electrode surfaces, but on arbitrary surfaces, using negative‐dielectrophoresis (n‐DEP). First, electrodes were designed and electric field simulations were performed to manipulate microparticles toward target areas. Based on the simulation results, multilayered array and grid (MLAG) electrodes, consisting of array electrodes surrounded by insulated regions and a grid electrode, were fabricated for the formation of localized, non‐uniform electric fields. The MLAG electrode was mounted to a target substrate in a face‐to‐face configuration with a spacer. When an AC voltage (4.60 V_rms and 1 MHz) was applied to the MLAG electrode, array patterns of 6 and 20 µm diameter microparticles were rapidly fabricated on the target substrate with ease. The results suggest that MLAG electrodes can be widely applied for the fabrication of biochips including cell arrays. Biotechnol. Bioeng. 2009; 104: 709–718 © 2009 Wiley Periodicals, Inc.     

NMR of fd coat protein

The conformations of the major coat protein of a filamentous bacteriophage can be described by nuclear magnetic resonance spectroscopy of the protein and the virus. The NMR experiments involve detection of the ¹³C and ¹H nuclei of the coat protein. Both the ¹³C and ¹H nuclear magnetic resonance (NMR) spectra show that regions of the polypeptide chain have substantially more motion than a typical globular protein. The fd coat protein was purified by gel chromatography of the SDS solubilized virus. Natural abundance ¹³C NMR spectra at 38 MHz resolve all of the nonprotonated aromatic carbons from the three phenylalanines, two tyrosines, and one tryptophan of the coat protein. The α carbons of the coat protein show at least two different classes of relaxation behavior, indicative of substantial variation in the motion of the backbone carbons in contrast to the rigidity of the α carbons of globular proteins. The ¹H spectrum at 360 MHz shows all of the aromatic carbons and many of the amide protons. Titration of a ¹H spectra gives the pKas for the tyrosines.     

Salt induced deprotonation of initially salt-free transfer RNA.

¹H NMR double resonance studies of valinomycin in (CD₃)₂SO were conducted at 90 MHz (FT-mode) and 250 MHz (correlation mode) to determine the mechanism of intramolecular nuclear Overhauser effects (NOE). These studies set specific constraints on any model for the conformation of valinomycin in (CD₃)₂SO and illustrate that NOE experiments of this type hold considerable promise for conformational studies of peptides, proteins and other biomolecules. The NOE's are positive at the lower frequency and negative at the higher frequency. Consideration of the theoretical dependence of the NOE on the proton-proton internuclear correlation time and on the resonance frequency indicates that these results are explained by a predominantly dipolar relaxation mechanism.     

Dielectrophoretic spectra of single cells determined by feedback-controlled levitation.

In this paper we have utilized the principle of dielectrophoresis (DEP) to develop an apparatus to stably levitate single biological cells using a digital feedback control scheme. Using this apparatus, the positive DEP spectra of both Canola plant protoplast and ligament fibroblast cells have been measured over a wide range of frequencies (1 kHz to 50 MHz) and suspending medium conductivities (11-800 muS/cm). The experimental data thus obtained have been interpreted in terms of a simple spherical cell model. Furthermore, utilizing such a model, we have shown that various cellular parameters of interest can be readily obtained from the measured DEP levitation spectrum. Specifically, the effective membrane capacitance of single cells has been determined. Values of 0.47 +/- 0.03 muF/cm2 for Canola protoplasts and 1.52 +/- 0.26 muF/cm2 for ligament fibroblasts thus obtained are consistent with those determined by other existing electrical methods.     

Poxvirus Antigen Staining of Immune Cells as a Biomarker to Predict Disease Outcome in Monkeypox and Cowpox Virus Infection in Non-Human Primates

Infection of non-human primates (NHPs) such as rhesus and cynomolgus macaques with monkeypox virus (MPXV) or cowpox virus (CPXV) serve as models to study poxvirus pathogenesis and to evaluate vaccines and anti-orthopox therapeutics. Intravenous inoculation of macaques with high dose of MPXV (>1–2×10⁷ PFU) or CPXV (>10² PFU) results in 80% to 100% mortality and 66 to 100% mortality respectively. Here we report that NHPs with positive detection of poxvirus antigens in immune cells by flow cytometric staining, especially in monocytes and granulocytes succumbed to virus infection and that early positive pox staining is a strong predictor for lethality. Samples from four independent studies were analyzed. Eighteen NHPs from three different experiments were inoculated with two different MPXV strains at lethal doses. Ten NHPs displayed positive pox-staining and all 10 NHPs reached moribund endpoint. In contrast, none of the three NHPs that survived anticipated lethal virus dose showed apparent virus staining in the monocytes and granulocytes. In addition, three NHPs that were challenged with a lethal dose of MPXV and received cidofovir treatment were pox-antigen negative and all three NHPs survived. Furthermore, data from a CPXV study also demonstrated that 6/9 NHPs were pox-antigen staining positive and all 6 NHPs reached euthanasia endpoint, while the three survivors were pox-antigen staining negative. Thus, we conclude that monitoring pox-antigen staining in immune cells can be used as a biomarker to predict the prognosis of virus infection. Future studies should focus on the mechanisms and implications of the pox-infection of immune cells and the correlation between pox-antigen detection in immune cells and disease progression in human poxviral infection.     

Continuous dielectrophoretic separation of cell mixtures

Use of stream-centered dielectrophoresis (1–4) produced continuous separations on three cell mixtures (1)Chlorella vulgaris withNetrium digitus, (2)Ankistrodesmus falcatus withStaurastrum gracile, and (3)Saccharomyces cerevisiae withNetrium digitus. Maximal separations were obtained for these mixtures of live cells at 100 kHz, 600 kHz, and 2.0 MHz, respectively. The technique was restricted to a frequency range of 0.01–32 MHz, and to suspensions of low conductivity in which microorganisms such as these algae and yeast are tolerant. Extension, however, to cellular organisms requiring higher osmolarity is readily feasible through the use of nonionic solutes such as sucrose, mannose, glycine, etc.     

Homokaryons from animal and plant cells generated by electrofusion

A new apparatus was constructed which enables the use of the electrofusion method to obtain polynuclear cells of various mammalian cell lines, erythrocytes and plant protoplasts. This technique was applied to both suspensions and monolayers. Electrical and other physical parameters were monitored to find optimal conditions for mutual contact of cells (dielectrophoresis) and subsequent fusion. In the suspension technique, dielectrophoresis of mouse erythrocytes occurred at a field frequency of 20 kHz and a strength of 500 V.cm-1, whereas cultured mammalian cells and plant protoplasts required a frequency of 1-1.4 MHz and a strength of 250-800 V.cm-1. Fusion of cells was induced after the application of 1 to 10 high-voltage pulses of 1-5 kV.cm-1, 10-36 microseconds duration. After these high-voltage pulses were to the monolayer of mouse L cells, about 12% viable homokaryons were obtained.     

Comparative effects of fusion facilitators on electrofusion attributes of N. tabacum mesophyll protoplasts

Mesophyll protoplasts isolated from in vitro-grown Nicotiana tabacum L. shoots were subjected to electrofusion.Dielectrophoresis was induced by an AC field of 50 V cm^-1 inter-electrode distance and 0.5 MHz oscillation frequency. Fusion was effected by two 0.7 kV cm^-1 DC pulses, each of 50 s duration, applied within one second of each other. Various chemical treatments were tested for their effects on dielectrophoresis efficiencies (percentages of protoplasts that made contact with at least one other protoplast under the AC field), fusion efficiencies (percentages of protoplasts participating in fusion events), cell lysis (percentages of protoplasts bursting during the electrofusion processes), overall viabilities of fusion products 24 h post-fusion and overall plating efficiencies 7 d post-fusion (percentages of fusion-derived cells that had undergone division). The various attributes assessed on the electrofusion of protoplasts in the control treatment, 10% mannitol, differed considerably for experiments carried out on different days. Relative to the control treatment, only the Ca²⁺ treatments, and to a lesser extent lipase treatment reduced dielectrophoresis efficiencies. Polyamines, cytochalasins and Ca²⁺ treatments significantly reduced cell lysis percentages. All electrofusion facilitators tested (except for spermine at 150 mg l^-1, the cytochalasins B and D, and Ca²⁺ treatments) increased fusion efficiencies to more than 1.5 times those obtained with the standard 10% mannitol electrofusion medium. Ca²⁺ treatments increased overall viabilities of fusion products by more than 1.5 times. With the exception of the prostaglandins, lecithin and CaCl₂ treatments, overall plating efficiencies were reduced by treatment of protoplasts with fusion facilitators. Substantial increases in overall plating efficiencies over those observed in the control treatment were obtained using prostaglandin F_2a, lecithin and CaCl₂.2H₂O treatments. The implications of the results are discussed.Abbreviations AC alternating current, approx.-approximately - BA benzylaminopurine, cv.-cultivar - DC direct current, diam.-diameter - FDA fluorescein diacetate - MS Murashige & Skoog (1962) - NAA napthaleneacetic acid - PCM protoplast culture medium - PIM protoplast isolation medium - PPM protoplast purification medium - rpm revolutions per minute - SD(n) standard deviation of a variate - SEM standard error of the mean     

Continuous dielectrophoretic separation of cell mixtures.

Use of stream-centered dielectrophoresis (1-4) produced continuous separations on three cell mixtures (1) Chorella vulgaris with Netrium digitus, (2) Ankistrodesmus falcatus with Staurastrum gracile, and (3) Saccharomyces cerevisiae with Netrium digitus. Maximal separations were obtained for these mixtures of live cells at 100 kHz, 600 kHz, and 2.0 MHz, respectively. The technique was restricted to a frequency range of 0.01-32 MHz, and to suspensions of low conductivity in which microorganisms such as these algae and yeast are tolerant. Extension, however, to cellular organisms requiring higher osmolarity is readily feasible through the use of nonionic solutes such as sucrose, mannose, glycine, etc.