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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 10×10 array of three-dimensional cell constructs. Some of these constructs, measuring ∼150 µm in diameter, 120 µm 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 10×10 array of cell constructs was compatible with that of a 1536 microtitre plate, and thus amenable to optical interrogation using automated plate reading equipment. [ABSTRACT FROM AUTHOR]

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 fxo 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 fxo values, ranging from 50 to 250 kHz, and as apoptosis progressed over 6 h, the upper value for fxo 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/m2, 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 fxo values does not agree with the earlier report that the fxo 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. [ABSTRACT FROM AUTHOR]

Membrane capacitance and membrane conductance values are reported for insulin secreting cells (primary β-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 β-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (106 cells per mL) on gold macrodisc electrodes and into human wound fluid (104 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 105 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]