Thomas Schnelle

Dielectric spectroscopy of single human erythrocytes at physiological ionic strength: dispersion of the cytoplasm.

Usually dielectrophoretic and electrorotation measurements are carried out at low ionic strength to reduce electrolysis and heat production. Such problems are minimized in microelectrode chambers. In a planar ultramicroelectrode chamber fabricated by semiconductor technology, we were able to measure the dielectric properties of human red blood cells in the frequency range from 2 kHz to 200 MHz up to physiological ion concentrations. At low ionic strength, red cells exhibit a typical electrorotation spectrum with an antifield rotation peak at low frequencies and a cofield rotation peak at higher ones. With increasing medium conductivity, both electrorotational peaks shift toward higher frequencies. The cofield peak becomes antifield for conductivities higher than 0.5 S/m. Because the polarizability of the external medium at these ionic strengths becomes similar to that of the cytoplasm, properties can be measured more sensitively. The critical dielectrophoretic frequencies were also determined. From our measurements, in the wide conductivity range from 2 mS/m to 1.5 S/m we propose a single-shell erythrocyte model. This pictures the cell as an oblate spheroid with a long semiaxis of 3.3 microns and an axial ratio of 1:2. Its membrane exhibits a capacitance of 0.997 x 10(-2) F/m2 and a specific conductance of 480 S/m2. The cytoplasmic parameters, a conductivity of 0.4 S/m at a dielectric constant of 212, disperse around 15 MHz to become 0.535 S/m and 50, respectively. We attribute this cytoplasmic dispersion to hemoglobin and cytoplasmic ion properties. In electrorotation measurements at about 60 MHz, an unexpectedly low rotation speed was observed. Around 180 MHz, the speed increased dramatically. By analysis of the electric chamber circuit properties, we were able to show that these effects are not due to cell polarization but are instead caused by a dramatic increase in the chamber field strength around 180 MHz. Although the chamber exhibits a resonance around 180 MHz, the harmonic content of the square-topped driving signals generates distortions of electrorotational spectra at far lower frequencies. Possible technological applications of chamber resonances are mentioned.

Three-dimensional electric field traps for manipulation of cells--calculation and experimental verification.

The forces acting on dielectric particles and living cells exposed to alternating and rotating fields generated by three-dimensional multi-electrode arrangements are investigated. Numerical procedures are described for the calculation of the electric field distribution and forces. The physical treatment considers electrodes of any shape and dielectric particles of complex structure. Particle and cell trapping are based on negative dielectrophoretic forces produced by high-frequency a.c. or rotating electric fields up to 400 MHz. Various multi-electrode systems were realised in commercially fabricated microelectrode systems, and tested for their ability to move and assemble microparticles or living cells without contact with the electrodes. The field distribution and accuracy of phase-controlled power application was tested using individual artificial particles trapped in the electric field cage. Position and trajectories of particle motion were measured. The paper gives an overview of electrode and field cage design in the microscale range.

Travelling wave-driven microfabricated electrohydrodynamic pumps for liquids

The basic principles of high-frequency travelling wave-driven micropumps are explained. We describe the design and construction of closed pump systems which contain planar and three dimensional components fabricated by semiconductor technology. Theoretical and experimental results and further perspectives are discussed.

Trapping of micrometre and sub-micrometre particles by high-frequency electric fields and hydrodynamic forces

We demonstrate that micrometre and sub-micrometre particles can be trapped, aggregated and concentrated in planar quadrupole electrode configurations by positive and negative dielectrophoresis. For particles less than in diameter, concentration is driven by thermal gradients, hydrodynamic effects and sedimentation forces. Liquid streaming is induced by the AC field itself via local heating and results, under special conditions, in vortices which improve the trapping efficiency. Microstructures were fabricated by electron-beam lithography and modified by UV laser ablation. They had typical gap dimensions between 500 nm and several micrometres. The theoretical and experimental results illustrate the basic principles of particle behaviour in ultra-miniaturized field traps filled with aqueous solutions. The smallest single particle that we could stably trap was a Latex bead of 650 nm. The smallest particles which were concentrated in the central part of the field trap were 14 nm in diameter. At high frequencies (in the megahertz range), field strengths up to 56 MV can be applied in the narrow gaps of 500 nm. Further perspectives for microparticle and macromolecular trapping are discussed.

Cell manipulation and cultivation under a.c. electric field influence in highly conductive culture media

Extreme miniaturisation of electrodes enabled us to apply high-frequency electric fields (between 100 kHz and several hundred MHz) of field strengths up to 50 kV/m into cell suspensions of high conductivity (several S/m), such as original cell culture media. The active electrode areas were additionally decreased and modified by insulating the terminals and/or coating of the electrodes with thin dielectric layers. Micro scaled electrode structures were fabricated on glass or silicon wafers in semiconductor technology. It could theoretically and experimentally be shown that cells exhibit exclusively negative dielectrophoresis if suspended in highly conductive media. Therefore, they can be repulsed from surfaces by appropriate arrangements of electrodes and easily be manipulated in free solution. Adherently growing animal cells, like mouse fibroblasts (3T3, L929), were cultivated in Dulbeccos Modification of Eagles Medium (DMEM) or RPMI 1640 under permanent field application (frequency: 10 MHz, field strength: 50-100 kV/m).

Paired microelectrode system: dielectrophoretic particle sorting and force calibration

Abstract We describe a closed microsystem used for free-flow dielectrophoretic separation of particles and cells. Particles have been separated on the basis of size and cells separated from artificial particles. Dielectrophoretic forces were calculated numerically and compared with experimental and analytic results. While the influence of local heating is of minor importance under our experimental conditions, the consideration of liquid-channel interfaces gives rise to an remarkable increase of dielectrophoretic forces.

The potential of dielectrophoresis for single-cell experiments

In this article, a microfluidic system was developed for sorting and high-content analysis of single cells using dielectrophoresis. It makes high-resolution imaging of suspended cells feasible immediately before depositing them into a microtiter plate. The microfluidic devices contain three-dimensional arrangements of radio-frequency driven electrodes and are used to gently manipulate suspended cells in flow. Viability of sorted and deposited cells is comparable to a control group. The influence of RF electric fields is studied using (a) calcium flux measurements and membrane potential dependent fluorescence dye on suspended human cells and (b) cultivation of yeast trapped for several days in dielectric field cages driven at field strengths up to 100 kV/m and 7 MHz.

Dielectric spectroscopy of human erythrocytes: investigations under the influence of nystatin

When placed in rotating electric fields red blood cells show a typical electrorotation spectrum with antifield rotation in the lower and cofield rotation in the higher frequency range. Assuming a spherical cell geometry, however, dielectrical parameters were obtained that differ from those measured by independent methods. Dielectrophoresis and, in particular, electrorotation yielded lower membrane capacitance values than expected. Introduction of an ellipsoidal model with an axis ratio of 1:2 allowed a description that proved to be consistent with dielectrophoresis and electrorotation data. For control cells an internal conductivity of 0.535 S/m, a specific membrane capacitance of 0.82 x 10(-2) F/m2, and a specific conductance of 480 S/m2 were obtained. The first characteristic frequency (frequency of fastest antifield rotation) and the related rotation speed can be measured quite quickly by means of a compensation method. Thus it was possible to follow changes of dielectric properties on individual cells after nystatin application. Ionophore-membrane interaction caused cell shrinkage in parallel to a decrease of the first characteristic frequency and rotation speed. Analysis of data revealed a decrease of the internal conductivity that is not only caused by ion loss but also, to a large extent, by a strong increase of hindrance because of shrinkage. Ionophore-induced membrane permeabilities can be calculated from volume decrease as well as from electrorotational data. In no case can these permeabilities count for the high membrane-AC conductivity that is attributed to the band-3 anion exchanging protein. The membrane-AC conductance was found not to be decreased for cells in Donnan equilibrium, which had leaked out almost completely.

Dielectrophoretic manipulation of suspended submicron particles

Planar and three‐dimensional multi‐electrode systems with dimensions of 2 — 40 μm were fabricated by IC technology and used for trapping and aggregation of microparticles. To achieve negative dielectrophoresis (repelling forces) in aqueous solution, radiofrequency (RF) electric fields were used. Experimentally, particles down to 100 nm in diameter were enriched and trapped as aggregates in field cages and dielectrophoretic microfilters and observed using confocal fluorimetry. Theoretically, single particles with an effective diameter down to about 35 nm should be trappable in micron field cages. Due to the unavoidable Ohmic heating, RF electric fields can induce liquid streaming in extremely small channels (12 μm in height). This can be used for pumping and particle enrichment but it enhances Brownian motion and counteracts dielectrophoretic trapping. Combining Brownian motion with ratchet‐like dielectrophoretic forces enables the creation of Brownian pumps that could be used as sensitive separation devices for submicron particles if liquid pumping is avoided in smaller structures.

Trapping of viruses in high-frequency electric field cages

High-frequency electric field cages can stably trap cells and microparticles in aqueous media [1, 2]. Such cages can be made by semiconductor fabricat ion techniques and are not to be confused with electromagnetic field devices used for t rapping atomic and elementary particles [3]. The behavior of various dielectric microparticles in uniform and nonuniform a.c. electric fields was investigated by Pohl in the 1970s and discussed in his monograph [4]. The mot ion of individual cells in nonuniform a.c. fields, termed dielectrophoresis (DP), was studied in subsequent decades [5]. The force, F, acting on a spherical dielectric particle of radius, r, in a timeperiodic electric field, f , can be expressed as a dipole approximat ion by