Development of a PCR-free electrochemical point of care test for clinical detection of methicillin resistant Staphylococcus aureus (MRSA).

An MRSA assay requiring neither labeling nor amplification of target DNA has been developed. Sequence specific binding of fragments of bacterial genomic DNA is detected at femtomolar concentrations using electrochemical impedance spectroscopy (EIS). This has been achieved using systematic optimisation of probe chemistry (PNA self-assembled monolayer film on gold electrode), electrode film structure (the size and nature of the chemical spacer) and DNA fragmentation, as these are found to play an important role in assay performance. These sensitivity improvements allow the elimination of the PCR step and DNA labeling and facilitate the development of a simple and rapid point of care test for MRSA. Assay performance is then evaluated and specific direct detection of the MRSA diagnostic mecA gene from genomic DNA, extracted directly from bacteria without further treatment is demonstrated for bacteria spiked into saline (10(6) cells per mL) on gold macrodisc electrodes and into human wound fluid (10(4) cells per mL) on screen printed gold electrodes. The latter detection level is particularly relevant to clinical requirements and point of care testing where the general threshold for considering a wound to be infected is 10(5) cells per mL. By eliminating the PCR step typically employed in nucleic acid assays, using screen printed electrodes and achieving sequence specific discrimination under ambient conditions, the test is extremely simple to design and engineer. In combination with a time to result of a few minutes this means the assay is well placed for use in point of care testing.

Dielectrophoretic tweezer for isolating and manipulating target cells.

The ability to isolate and accurately position single cells in three dimensions is becoming increasingly important in many areas of biological research. The authors describe the design, theoretical modelling and testing of a novel dielectrophoretic (DEP) tweezer for picking out and relocating single target cells. The device is constructed using facilities available in most electrophysiology laboratories, without the requirement of sophisticated and expensive microfabrication technology, and offers improved practical features over previously reported DEP tweezer designs. The DEP tweezer has been tested using transfected HEI-193 human schwannoma cells, with visual identification of the target cells being aided by labelling the incorporated gene product with a green fluorescent protein.

Dielectrophoretic assembly of insulinoma cells and fluorescent nanosensors into three-dimensional pseudo-islet constructs.

Dielectrophoretic forces, generated by radio-frequency voltages applied to micromachined, transparent, indium tin oxide electrodes, have been used to condense suspensions of insulinoma cells (BETA-TC-6 and INS-1) into a 10 x 10 array of three-dimensional cell constructs. Some of these constructs, measuring approximately 150 microm in diameter, 120 microm in height and containing around 1000 cells, were of the same size and cell density as a typical islet of Langerhans. With the dielectrophoretic force maintained, these engineered cell constructs were able to withstand mechanical shock and fluid flow forces. Reproducibility of the process required knowledge of cellular dielectric properties, in terms of membrane capacitance and membrane conductance, which were obtained by electrorotation measurements. The ability to incorporate fluorescent nanosensors, as probes of cellular oxygen and pH levels, into these 'pseudo-islets' was also demonstrated. The footprint of the 10 x 10 array of cell constructs was compatible with that of a 1536 microtitre plate, and thus amenable to optical interrogation using automated plate reading equipment.

Dielectrophoretic detection of membrane morphology changes in Jurkat T-cells undergoing etoposide-induced apoptosis.

A dielectrophoresis (DEP) cell profiler, with concurrent FACS measurements, was used to monitor the morphological changes of Jurkat T-cells as they progressed through chemically induced apoptosis using etoposide. The cell 'physiometry' profiling technique measures the radius and the so-called DEP crossover frequency f(xo) of individual cells in a suspension, and this information was used to determine the effective plasma membrane capacitance of each cell. Control cells (n=526) exhibited a dynamic spread of f(xo) values, ranging from 50 to 250 kHz, and as apoptosis progressed over 6 h, the upper value for f(xo) progressively increased and extended beyond 500 kHz. This corresponded to a reduction in plasma membrane capacitance from 13.34 (+/-2.88) to 10.49 (+/-4.00) mF/m(2), and reflected a general smoothing of the membrane through loss of microvilli, for example. This is in broad agreement with previously reported studies of HL-60 cells undergoing apoptosis, but the authors' observation of a dynamic spread of f(xo) values does not agree with the earlier report that the f(xo) values for viable and apoptotic cells fall into two separable, relatively narrow, frequency bands. This has implications when devising protocols for the efficient DEP separation of viable, apoptotic and necrotic cells.

Controlling cell destruction using dielectrophoretic forces.

Measurements are reported of the main factors, namely the AC voltage frequency and magnitude, that were observed to influence the number of cells destroyed during dielectrophoresis (DEP) experiments on Jurkat T cells and HL60 leukemia cells. Microelectrodes of interdigitated and quadrupolar geometries were used. A field-frequency window has been identified that should be either avoided or utilised, depending on whether or not cell damage is to be minimised or is a desired objective. The width and location of this frequency window depends on the cell type, as defined by cell size, morphology and dielectric properties, and is bounded by two characteristic frequencies. These frequencies are the DEP cross-over frequency, where a cell makes the transition from negative to positive DEP, and a frequency determined by the time constant that controls the frequency dependence of the field induced across the cell membrane. When operating in this frequency window, and for the microelectrode designs used in this work, cell destruction can be minimised by ensuring that cells are not directed by positive DEP to electrode edges where fields exceeding 30-40 kV/m are generated. Alternatively, this field-frequency window can be exploited to selectively destroy specific cell types in a cell mixture.

Electrokinetic measurements of membrane capacitance and conductance for pancreatic beta-cells.

Membrane capacitance and membrane conductance values are reported for insulin secreting cells (primary -cells and INS-1 insulinoma cells), determined using the methods of dielectrophoresis and electrorotation. The membrane capacitance value of 12.57 (+/-1.46) mFm(-2), obtained for -cells, and the values from 9.96 (+/-1.89) mFm(-2) to 10.65 (+/-2.1) mFm(-2), obtained for INS-1 cells, fall within the range expected for mammalian cells. The electrorotation results for the INS-1 cells lead to a value of 36 (+/-22) Sm(-2) for the membrane conductance associated with ion channels, if values in the range 2-3 nS are assumed for the membrane surface conductance. This membrane conductance value falls within the range reported for INS cells obtained using the whole-cell patch-clamp technique. However, the total 'effective' membrane conductance value of 601 (+/-182) Sm(-2) obtained for the INS-1 cells by dielectrophoresis is significantly larger (by a factor of around three) than the values obtained by electrorotation. This could result from an increased membrane surface conductance, or increased passive conduction of ions through membrane pores, induced by the larger electric field stresses experienced by cells in the dielectrophoresis experiments.

Viability of Giardia intestinalis cysts and viability and sporulation state of Cyclospora cayetanensis oocysts determined by electrorotation.

Electrorotation is a noninvasive technique that is capable of detecting changes in the morphology and physicochemical properties of microorganisms. Electrorotation studies are reported for two intestinal parasites, Giardia intestinalis and Cyclospora cayetanensis. It is concluded that viable and nonviable G. intestinalis cysts can be differentiated by this technique, and support for this conclusion was obtained using a fluorogenic vital dye assay and morphological indicators. The viability of C. cayetanensis oocysts (for which no vital dye assay is currently available) can also be determined by electrorotation, as can their sporulation state. Modeling of the electrorotational response of these organisms was used to determine their dielectric properties and to gain an insight into the changes occurring within them. Electrorotation offers a new, simple, and rapid method for determining the viability of parasites in potable water and food products and as such has important healthcare implications.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.