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Biomedical subjects

R Pethig

Publications and source records attributed to R Pethig.

At least 19 recordsLinked to original sources
PubMed1998

Manipulation of herpes simplex virus type 1 by dielectrophoresis.

The frequency-dependent dielectrophoretic behaviour of an enveloped mammalian virus, herpes simplex virus type 1 is described. It is demonstrated that over the range 10 kHz-20 MHz, these viral particles, when suspended in an aqueous medium of conductivity 5 mS m(-1), can be manipulated by both positive and negative dielectrophoresis using microfabricated electrode arrays. The observed transition from positive to negative dielectrophoresis at frequencies around 4.5 MHz is in qualitative agreement with a simple model of the virus as a conducting particle surrounded by an insulating membrane.

Biophysics↗
PubMed1998

Electrorotation and dielectrophoresis.

Using microelectrode structures, various forms of electric fields, such as non-uniform, rotating and travelling wave, can be imposed on particles of sizes ranging from proteins and viruses to micro-organisms and cells. Each type of particle responds to the forces exerted on them in a unique way, allowing for their controlled and selective manipulation as well as their characterization. Moreover, particles of the same type but of different viability can be distinguished in a simple, reliable manner. This review outlines the principles that govern the way in which bioparticles respond to these various field types, and how they can be exploited. Examples of current and potential biotechnological and biomedical applications are given, along with a critical comparison of current techniques.

Animals↗
PubMed1998

Future trends in diagnosis using laboratory-on-a-chip technologies.

There has been an enormous growth in the development of biotechnological applications, where advances in the techniques of microelectronic fabrication and the technologies of miniaturization and integration in semiconductor industries are being applied to the production of Laboratory-on-a-Chip devices. The aim of this development is to create devices that will perform the same processes that are currently carried out in the laboratory in reduced timescales, at a lower cost, requiring less reagents, and with a greater resolution of detection and specificity. The expectations of this Laboratory-on-a-Chip revolution is that this technology will facilitate rapid advances in gene discovery, genetic mapping and gene expression with broader applications ranging from infectious diseases and cancer diagnostics to food quality and environmental testing. A review of the current state of development in this field reveals the scale of the ongoing revolution and serves to highlight the advances that can be perceived in the development of Laboratory-on-a-Chip technologies. Since miniaturization can be applied to such a wide range of laboratory processes, some of the sub-units that can be used as building blocks in these devices are described, with a brief description of some of the fabrication processes that can be used to create them.

Animals↗
PubMed1998

Automatic cell electrorotation measurements: studies of the biological effects of low-frequency magnetic fields and of heat shock.

A computer-aided automatic imaging technique has been developed for measuring the electrorotation spectra of up to 256 particles at the same time. This offers advantages over the conventional manual method, especially when rapidly acquired statistical data are necessary in investigations of the response of cells or test beads to chemical exposure, for example. We have applied this technique to investigate the biological effects of heat shock and low-frequency EM fields reported by others for yeast cells. Although heat shock effects were observed, no changes of the electrorotational behaviour could be detected after exposing the cells to 50 Hz, 8 and 80 microT fields. Although this does not rule out the possibility that the cells were influenced by the magnetic fields, it does limit the number of possible physicochemical changes that might have occurred to their cell walls and membranes.

Automation↗
PubMed1997

Electrorotation of liposomes: verification of dielectric multi-shell model for cells.

The reliability of multi-shell dielectric models, used to describe the ac electrokinetic behaviour of cells, has been tested by performing electrorotation and dielectrophoretic measurements on unilamellar, oligolamellar, and multilamellar liposomes of diameters ranging from 5 to 24 microm. Fluorescence microscopy, flow cytometry and electron spin resonance were used to characterise the morphology and membranes of the liposomes. The dielectric properties of the various types of liposomes, based on appropriate dielectric shell models, were then analysed using a general purpose, recursive, algorithm. Through simulations, the confidence levels that can be assigned to parameters derived through application of simple shell models are estimated. From this, we confirm that electrorotation data enable accurate determinations to be made of the dielectric properties of the outermost membrane of liposomes, and provide good indications of the level of complexity of the shells and internal compartments. We also demonstrate that, used with sufficient additional information, such as that provided by dielectrophoresis, electrorotation data yields unique solutions for the dielectric parameters of liposome-like particles.

Electric Conductivity↗
PubMed1997

Dielectrophoretic detection of changes in erythrocyte membranes following malarial infection.

The dielectric properties of normal erythrocytes were compared to those of cells infected with the malarial parasite Plasmodium falciparum. Normal cells provided stable electrorotation spectra which, when analyzed by a single-shelled oblate spheroid dielectric model, gave a specific capacitance value of 12 +/- 1.2 mF/m2 for the plasma membrane, a cytoplasmic permittivity of 57 +/- 5.4 and a cytoplasmic conductivity of 0.52 +/- 0.05 S/m. By contrast, parasitized cells exhibited electrorotation spectra with a time-dependency that suggested significant net ion outflux via the plasma membrane and it was not possible to derive reliable cell parameter values in this case. To overcome this problem, cell membrane dielectric properties were instead determined from dielectrophoretic crossover frequency measurements made as a function of the cell suspending medium conductivity. The crossover frequency for normal cells depended linearly on the suspension conductivity above 20 mS/m and analysis according to the single-shelled oblate spheroid dielectric model yielded values of 11.8 mF/m2 and 271 S/m2, respectively, for the specific capacitance and conductance of the plasma membrane. Unexpectedly, the crossover frequency characteristics of parasitized cells at high suspending medium conductivities were non-linear. This effect was analyzed in terms of possible dependencies of the cell membrane capacitance, conductance or shape on the suspension medium conductivity, and we concluded that variations in the membrane conductance were most likely responsible for the observed non-linearity. According to this model, parasitized cells had a specific membrane capacitance of 9 +/- 2 mF/m2 and a specific membrane conductance of 1130 S/m2 that increased with increasing cell suspending medium conductivity. Such conductivity changes in parasitized cells are discussed in terms of previously observed parasite-associated membrane pores. Finally, we conclude that the large differences between the dielectrophoretic crossover characteristics of normal and parasitized cells should allow straightforward sorting of these cell types by dielectrophoretic methods.

Animals↗
PubMed1997

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.

Animals↗
PubMed1996

Dielectrophoretic separation of bacteria using a conductivity gradient.

Dielectrophoresis, the lateral motion induced on particles by non-uniform electric fields, is a sensitive function of the electrical conductivity of the particle suspending medium. This dependence is exploited in a new technique for separating bioparticles from suspended mixtures. The bioparticles are first immobilised by positive dielectrophoresis at electrodes in a separation chamber, and the conductivity of the liquid flowing through the chamber is then gradually and continuously increased so as to produce a conductivity gradient with time. The bioparticles are released from the electrodes according to their own dielectric properties and as a function of flow rate and medium conductivity. This is demonstrated for pure suspensions and mixtures of the bacteria Bacillus subtilis, Escherichia coli and Micrococcus luteus.

Bacillus subtilis↗
PubMed1996

Effect of biocide concentration on electrorotation spectra of yeast cells.

The effect of the biocide Cosmocil (polyhexanide) at different concentrations on the electrorotation spectra of yeast cells is investigated over the frequency range from 1 kHz to 10 MHz. The dielectric properties of the yeast, before and after biocide treatment, were deduced from the electrorotation spectra using two-shell ellipsoid modelling methods that have been well tested for other heterogeneous biological systems. The results show a gradual increase in the cytoplasmic membrane conductivity with increasing biocide concentration, rather than an "all-or-nothing' breakdown of the membrane. The technique gives a quantitative analysis of the toxic damage by chemicals to cells and can be exploited in the development of new pharmacological agents.

Biguanides↗
PubMed1996

The dielectrophoresis enrichment of CD34+ cells from peripheral blood stem cell harvests.

There is considerable interest in isolating the CD34+ cell population from leukaemic patients undergoing peripheral blood stem cell harvests. The techniques currently available make use of antibodies specific to the CD34+ surface markers. However, all of these techniques involve disturbance of the cell surface, are time-consuming and relatively expensive. In this study, we have used dielectrophoresis, which does not rely on the presence of cell-specific markers, to separate CD34+ cells from peripheral blood stem cell harvest samples containing an untreated natural mixed cell population. The separation is achieved by exploiting differences in the inherent dielectric properties of the various cell types. Samples obtained from peripheral blood stem cell harvests were resuspended in medium with a conductivity of less than 50 microS/cm and introduced into the dielectrophoretic separation chamber. Alternating field frequencies, from 500 kHz to 5 kHz, were used to collect cell fractions which were analysed by FACS, using a CD34-specific antibody, to quantify the CD34+ population within the fractions. On average a nearly five-fold increase in the frequency of the CD34+ cell population was observed in the fractions collected within the 50-10 kHz range. For this dielectrophoretic separation technique to be suitable in harvesting CD34+ cells for transplantation, it is important to demonstrate that the cells remain viable after the separation process. Cells obtained from each fraction grew when plated in colony assay cultures, GM-CFU and BFU-E, demonstrating that the cells remain normal, viable and capable of colony formation when cultured for 2 weeks. The number of colonies formed correlated with the percentage of CD34+ cells in each fraction. The dielectrophoretic separation technique is simple to operate, the separation is fast, the procedure non-invasive and although not tested has the potential to be incorporated as a batch-wise online facility with the standard harvesting equipment to increase the yield and speed of CD34+ cells in the PBSC harvest.

Antigens, CD34↗
PubMed1996

Assays for microbial contamination and DNA analysis based on electrorotation.

A new assay is described for determining the concentration and viability of waterborne micro-organisms. The method exploits the fact that when a latex bead coated with a specific binding agent is complexed to the target analyte, the analyte complex formed assumes new dielectric properties that can be monitored by its electrorotation response. This generic technology is applicable to a wide range of organism and toxicological diagnostic tests, as well as to other areas of biotechnology. An example is given of its possible application to DNA sequence analysis.

Animals↗
PubMed1995

Differentiation of viable and non-viable bacterial biofilms using electrorotation.

A new technique for studying the properties of biofilms has been developed, based on the phenomenon of electrorotation. Biofilms of Klebsiella rubiacearum were formed on the surfaces of 6 microns diameter polystyrene beads, and the presence of such films was found to alter their electrorotation spectra. The effects of adding a biocide (polyhexanide) to the surrounding aqueous medium was also investigated. The dielectric properties of the beads with biofilms, before and after biocide treatment, were interpreted from the electrorotation spectra using modelling methods that have been well tested for other heterogeneous biological systems. The technique is of value in understanding the physico-chemical properties of biofilms and can be adapted for monitoring the presence of toxic chemicals and for testing the activity of biocides against biofilms.

Biofilms↗
PubMed1995

Separation of human breast cancer cells from blood by differential dielectric affinity.

Electrorotation measurements were used to demonstrate that the dielectric properties of the metastatic human breast cancer cell line MDA231 were significantly different from those of erythrocytes and T lymphocytes. These dielectric differences were exploited to separate the cancer cells from normal blood cells by appropriately balancing the hydrodynamic and dielectrophoretic forces acting on the cells within a dielectric affinity column containing a microelectrode array. The operational criteria for successful particle separation in such a column are analyzed and our findings indicate that the dielectric affinity technique may prove useful in a wide variety of cell separation and characterization applications.

Blood↗
PubMed1995

Dielectrophoretic separation and enrichment of CD34+ cell subpopulation from bone marrow and peripheral blood stem cells.

Dielectrophoresis has been used to enrich selected cell subpopulations in a mixed cell population by exploiting differential dielectric properties. Six-fold enrichment of stem cells expressing the CD34+ antigen has been achieved for bone marrow samples and peripheral blood, without the requirement for initial chemical treatment associated with immunoadsorption techniques.

Antigens, CD↗
PubMed1994

Changes in Friend murine erythroleukaemia cell membranes during induced differentiation determined by electrorotation.

We used electrorotation measurements to investigate alterations in the plasma membranes of DS19 murine erythroleukaemia cells that accompanied erythropoietic differentiation induced by hexamethylene bisacetamide (HMBA). Following 3 days of HMBA treatment, the mean cell membrane specific capacitance determined from electrorotation spectra of individual, viable cells at physiological tonicity (300 mosmol/kg) fell from 1.74 to 1.53 microF/cm2, in agreement with trends observed earlier by dielectrophoretic measurements on bulk cell populations. Scanning and transmission electron microscopy revealed that the relatively high values found for cell membrane capacitance (> 1 microF/cm2) reflected the large area of plasma membrane associated with complex surface morphology including numerous microvilli. Furthermore, it demonstrated that the fall in membrane capacitance during HMBA treatment correlated with a reduction in the density of these complex surface features. Differences in the mechanical characteristics of the cell membranes of untreated and treated cells were then examined by exposing cells to osmotic stress. The intricacy of membrane morphology intensified with increasing osmolality of the suspending medium and this was reflected in higher specific capacitance values. When the osmolality was increased from 210 to 450 mosmol/kg, the mean membrane capacitance of untreated DS19 cells changed from 1.58 to 2.05 microF/cm2 while that for HMBA-treated cells changed from 1.47 to 1.72 microF/cm2, a significantly smaller response. This demonstrated that cells exposed to 72 h of differentiation treatment had an enhanced mechanical resilience as compared with their untreated counterparts, evidencing the early stages of the development of the membrane skeleton which becomes fully developed in mature erythrocytes. Our findings demonstrate the value of electrorotation measurements as a method for the non-invasive characterisation of viable leukaemic cells and their responses to stimuli and show that the membrane capacitance values so derived reflect membrane morphology.

Acetamides↗
PubMed1994

Separation of viable and non-viable yeast using dielectrophoresis.

Dielectrophoresis, the movement of particles in non-uniform AC electric fields, was used to rapidly separate viable and non-viable yeast cells with good efficiency. Known mixtures of viable and heat-treated cells of Saccharomyces cerevisiae were separated and selectively isolated using positive and negative dielectrophoretic forces generated by microelectrodes in a small chamber. Good correlations with the initial known relative compositions were obtained by direct microscopic counting of cells at the electrodes after initial dielectrophoretic separation (r = 0.995), from methylene blue staining (r = 0.992) and by optical absorption measurements (r = 0.980) of the effluent after selectively flushing out the viable and non-viable cells from the chamber. Through measurement of cell viability by staining with methylene blue and plate counts, for an initial suspension of approx. 1.4 x 10(7) cells per ml containing 60% non-viable cells, the dielectrophoretically separated non-viable fraction contained 3% viable cells and the viable fraction 8% dead cells. The separation efficiency is increased by dilution of the initial suspension or by repeat operation(s). Cell viability was not affected by the separation procedure.

Electrophoresis↗
PubMed1993

Membrane changes accompanying the induced differentiation of Friend murine erythroleukemia cells studied by dielectrophoresis.

Dielectrophoresis measurements obtained using an image processing technique are reported over the frequency range 1 Hz to 100 kHz for the Friend murine erythroleukemia cell lines DS19 and R1 before and after treatment with hexamethylene bisacetamide and dimethylsulfoxide, agents that induce terminal differentiation in DS19 but not in R1 cells. Data are analyzed according to the single shell dielectric model of the cell. The membrane capacitance was found to fall by 30% and membrane conductivity by a factor of at least 5 when DS19 cells were induced to differentiate. R1 cells showed no such response. While the theoretical model was found to be useful for comparing differences in data for the different cell lines, several significant discrepancies between its predictions and the experimental data were observed, including positive dielectrophoretic collection at frequencies below 20 Hz and a smaller than predicted response to the membrane permeabilizing agents saponin and valinomycin. Factors that may have accounted for these discrepancies include surface charge effecgs, conduction parallel to the plasma membrane surface, and intracellular compartments.

Cell Differentiation↗