Günter Fuhr

A 3-D microelectrode system for handling and caging single cells and particles

Abstract We have designed and constructed several designs of 3-D microelectrode systems consisting of two layers of electrode structures separated by a 40 μm thick polymer spacer forming a flow channel. The electrode elements such as funnel, aligner, cage and switch are driven by alternating current (AC) and/or rotating electric fields. They are designed to focus, trap and separate eukaryotic cells (Jurkat) or latex particles with a diameter of 10–30 μm using negative dielectrophoresis (nDEP). The electrode width is ∼10 μm and active electrode surfaces have been minimised in order to reduce heating of the solution. A more flexible employment of the different electrode elements was realised by working with up to three generators, and/or a laboratory-made distribution box. The electrodes of the funnel, aligner or switch were operated with 5–11 V at 5–15 MHz, efficient handling of particles could be achieved with flow rates up to 3500 μm/s. Cells could be aligned effectively at flow rates up to 300 μm/s in PBS. Latex particles could be retained within the dielectric field cages (DFC) or aligner against a laminar flow of 40–200 μm/s using an amplitude of 8 V at 5–15 MHz. Cells could be held at flow rates up to 50 μm/s. Numerical calculations for dielectric forces and the induced membrane potential in field cages are given for solutions of different conductivities at different applied frequencies.

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.

Microfabricated electrohydrodynamic (EHD) pumps for liquids of higher conductivity

A traveling-wave-driven electrohydrodynamic micropump without moving parts is discussed. The fundamental operating principles, such as high-frequency traveling waves, a self-stabilizing temperature gradient, and increased wave number, are outlined. The main advantages of the realized pump are its ability to move conductive liquids such as water and weak electrolyte solutions, the lack of any movable parts, and integration. A microfabricated structure demonstrating the pump operation is outlined, and quantitative results are described. Typical parameters characterizing the advantages and limitations of the pumping principle are discussed. Perspectives for optimization of the realized micropump can be seen in further miniaturization and increased number of electrodes. Possible applications are biological, medical, and chemical devices that can deliver accurately metered quantities of fluids in the nl/min and mu l/min range. >

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).

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.

Snow algae from northwest Svalbard: their identification, distribution, pigment and nutrient content

Abstract We mapped coloured snow during the summers of 1995 and 1996 at about 60 localities in the coastal region of northwest Spitsbergen. The colour was mainly induced by snow algae (Chlamydomonas spp. and Chloromonas spp.). In the late summer of 1996, snow algal fields of several hundred meters in size were observed along the west and north coasts. They had no preferred geographical orientation. We studied the abundance of primary pigments and secondary carotenoids from different developmental stages of the snow algae of Chlamydomonas spp. under natural conditions. Extensive accumulation of astaxanthin and its esters accompanied the transition from green biflagellated cells to orange spores, hypnozygotes and dark-red cysts. The photoprotective effect of the secondary carotenoids is enhanced by concentration in cytoplasmic lipid droplets around the nucleus and chloroplast. The nutrient content of melt-water and snow algae had no direct correlation with the content of secondary carotenoids. Relatively high Fe, Ca, P, K and Al contents of snow algae were found, suggesting a good supply of these mineral elements.

A Novel Class of Amitogenic Alginate Microcapsules for Long-Term Immunoisolated Transplantation

Abstract: In the light of results of clinical trials with immunoisolated human parathyroid tissue Ba2+‐alginate capsules were developed that meet the requirements for long‐term immunoisolated transplantation of (allogeneic and xenogeneic) cells and tissue fragments. Biocompatibility of the capsules was achieved by subjecting high‐M alginate extracted from freshly collected brown algae to a simple purification protocol that removes quantitatively mitogenic and cytotoxic impurities without degradation of the alginate polymers. The final ultra‐high‐viscosity, clinical‐grade (UHV/CG) product did not evoke any (significant) foreign body reaction in BB rats or in baboons. Similarly, the very sensitive pERK assay did not reveal any mitogenic impurities. Encapsulated cells also exhibited excellent secretory properties under in vitro conditions. Despite biocompatible material, pericapsular fibrosis is also induced by imperfect capsule surfaces that can favor cell attachment and migration under the release of material traces. This material can interact with free end monomers of the alginate polymers under formation of mitogenic advanced glycation products. Smooth surfaces, and thus topographical biocompatibility of the capsules (visualized by atomic force microscopy), can be generated by appropriate crosslinking of the UHV/CG‐alginate with Ba2+ and simultaneous suppression of capsule swelling by incorporation of proteins and/or perfluorocarbons (i.e., medically approved compounds with high oxygen capacity). Perfluorocarbon‐loaded alginate capsules allow long‐term non‐invasive monitoring of the location and the oxygen supply of the transplants by using 19F‐MRI. Transplantation studies in rats demonstrated that these capsules were functional over a period of more than two years.