Gerard H. Markx

Applications of dielectrophoresis in biotechnology.

Recent progress in the development of microelectrode structures has led to new techniques for the dielectrophoretic characterization and sorting of cells, microorganisms and other bioparticles using nonuniform AC electric fields. These methods utilize differences in the dielectric polarizabilities of cells for their effectiveness, and factors controlling such properties include the conductivity and permittivity of membranes and any cell walls, electrical double layers associated with surface charges, cell morphologies, and internal structures. Applications of dielectrophoresis have included the selective spatial manipulation and separation of mixtures of bacteria, viable and unviable cells, cancerous and normal cells, and red and white blood cells.

The dielectric properties of biological cells at radiofrequencies: applications in biotechnology

The study of the dielectric properties of cells in the radiofrequencies is increasingly leading to new practical applications, including online techniques for biomass measurements and novel techniques for the electrokinetic separation, manipulation, and characterization of single cells. In this review, we will discuss the dielectric properties of cells and their components and the electrical techniques that use them. This will be done mainly in the context of biotechnology but some applications in medicine will also be highlighted.

Dielectrophoretic characterization and separation of micro-organisms

The effective electrical conductivity values for a variety of Gram-negative and positive bacteria have been determined in the frequency range 10 kHz to 100 kHz. This information enabled experimental conditions to be selected where micro-organisms of different species can be separated using dielectrophoresis - the movement of particles induced by non-uniform AC electric fields. Mixtures of micro-organisms of different species were separated locally on a microscope slide using micro-electrodes of polynomial geometry, and their physical isolation into two separate suspensions was accomplished using a dielectrophoresis chamber that incorporated interdigitated, castellated micro-electrodes.

Selective dielectrophoretic confinement of bioparticles in potential energy wells

The potential energy surfaces generated by microelectrodes of polynomial and interdigitated castellated geometry have been calculated for particles experiencing both positive and negative dielectrophoretic forces. The resulting forms of particle collection at these electrodes are governed by the locations of the potential energy wells, and the theoretical predictions of the modes of collection for particles experiencing positive and negative dielectrophoretic forces are verified using mixtures of viable and non-viable yeast cells, as well as of bacteria and blood cells. An important result for the interdigitated electrodes is the finding that particles trapped in potential energy wells under the action of negative dielectrophoresis can be more easily removed from the electrode structure (e.g. by fluid flow or gravitational forces) than those trapped under positive dielectrophoresis. This was verified for mixtures of bacteria and blood cells, viable and non-viable yeast cells.

DEP-FFF: Field-Flow Fractionation Using Non-Uniform Electric Fields

Dielectrophoresis (DEP) - the movement of particles in non-uniform electric fields - can be used in combination with Field Flow Fractionation (FFF) to separate particles with differing dielectric properties. An introduction is given to the technique of DEP-FFF and its application to the separation of cells and other particles. The separation of yeast cells using the subtechniques of steric and hyperlayer DEP-FFF is demonstrated. It is shown that the hyperlayer-DEP-FFF techniques have a number of advantages, including an improved separation efficiency and reduced adhesion to chamber walls. The hyperlayer-DEP-FFF separation technique is also independent of particle size and allows the use of higher medium conductivities than for conventional DEP methods.

The permittistat: a novel type of turbidostat

Summary: Bakers yeast was grown in a novel type of turbidostat in which the steady-state biomass level was controlled not by the optical turbidity but by the dielectric permittivity of the suspension at appropriate radio frequencies. Dry weight, fresh weight, the optical density at 600 nm, percentage viability (from methylene blue staining), bud count and ethanol concentration were measured off-line and the cell size distribution was recorded using flow cytometry. Any changes in the physiological properties of the yeast had a negligible effect on the ratio between the permittivity set (and measured) and the steady-state dry weight, fresh weight or optical density of the cultures. The permittistat was found to provide an extremely convenient means for carrying out turbidostatic culture.

The use of electric fields in tissue engineering: A review.

The use of electric fields for measuring cell and tissue properties has a long history. However, the exploration of the use of electric fields in tissue engineering is only very recent. A review is given of the various methods by which electric fields may be used in tissue engineering, concentrating on the assembly of artificial tissues from its component cells using electrokinetics. A comparison is made of electrokinetic techniques with other physical cell manipulation techniques which can be used in the construction of artificial tissues.

Separation by dielectrophoresis of dormant and nondormant bacterial cells of Mycobacterium smegmatis

The dielectrophoretic behavior of active, dead, and dormant Mycobacterium smegmatis bacterial cells was studied. It was found that the 72-h-old dormant cells had a much higher effective particle conductivity (812+/-10 muS cm(-1)), almost double that of active cells (560+/-20 muS cm(-1)), while that of dead (autoclaved) M. smegmatis cells was the highest (950+/-15 muS cm(-1)) overall. It was also found that at 80 kHz, 900 muS cm(-1) dead cells were attracted at the edges of interdigitated castellated electrodes by positive dielectrophoresis, but dormant cells were not. Similarly, at 120 kHz, 2 muS cm(-1) active cells were attracted and dormant cells were not. Using these findings a dielectrophoresis-based microfluidic separation system was developed in which dead and active cells were collected from a given cell suspension, while dormant cells were eluted.

Formation of multilayer aggregates of mammalian cells by dielectrophoresis

The formation of aggregates of mammalian cells at interdigitated oppositely castellated electrodes by positive dielectrophoresis was investigated. It is shown that, by using a constant small flow of fresh sorbitol iso-osmotic buffer through the chamber to remove ions leaking from the cells, a high positive DEP force can be maintained throughout the formation of the aggregates. Flow-rate dependent optima were found in the aggregate height as a function of the electrode size. It is shown that at low flow rates the creation of aggregates of mammalian cells with heights over 150 µm is feasible using relatively low voltages (20 Vpk–pk, 1 MHz). The formation of layered aggregates of two specialized cell types—stromal cells and Jurkat T lymphocytes—is demonstrated. The work confirms that dielectrophoresis can be reliably used for the formation of aggregates with three-dimensional architectures, which could be used as artificial microniches for the study of interactions between cells.

Flow-through devices for the ac electrokinetic construction of microstructured materials

With the aid of computer simulation, flow-through devices have been devised for the continuous construction of microstructured materials using non-uniform ac electric fields. Particles can be concentrated and guided along channels defined by non-uniform electric fields generated between microelectrodes. The resulting streams of particles emanating from the microelectrode structures can subsequently be immobilized to form materials with particles embedded in defined locations. Experiments with latex beads with a diameter of 6 µm, suspended in high purity low-melting agarose at a concentration of 0.75% and temperatures over 60 °C, showed that a linear stream of particles can be created by the combined application of negative dielectrophoresis and hydrodynamic flow forces. By guiding the stream of particles onto a conveyor, it was then possible to create a continuous film of agarose containing latex beads in defined positions. Potential applications of the method in the creation of biomaterials such as tissues and biofilms are discussed.