On the low-frequency dispersion observed in dielectrophoresis spectra.

The foundation of dielectrophoresis (DEP) as a tool for biological investigation is the use of the Clausius-Mossotti (C-M) factor to model the observed behaviour of cells experiencing DEP across a frequency range. Nevertheless, it is also the case that at lower frequencies, the DEP spectrum deviates from predictions; there exists a rise in DEP polarisability, which varies in frequency and magnitude with different cell types and medium conductivities. In order to evaluate the origin of this effect, we have studied DEP spectra from five cell types (erythrocytes, platelets, neurons, HeLa cancer cells and monocytes) in several conditions including medium conductivity and cell treatment. Our results suggest the effect manifests as a low-pass dispersion whose cut-off frequency varies with membrane conductance and capacitance as determined using the DEP spectrum; the effect also varies as a logarithm of medium conductivity and Debye length. These together suggest that the values of membrane capacitance and conductance depend not only on the impedance of the membrane itself, but also of the surrounding double layer. The amplitude of the effect in different cell types compared to the C-M factor was found to correlate with the depolarisation factors for the cells' shapes, suggesting that this ratio may be useful as an indicator of cell shape for DEP modelling.

Circadian rhythmicity in murine blood: Electrical effects of malaria infection and anemia.

Circadian rhythms are biological adaptations to the day-night cycle, whereby cells adapt to changes in the external environment or internal physiology according to the time of day. Whilst many cellular clock mechanisms involve gene expression feedback mechanisms, clocks operate even where gene expression is absent. For example, red blood cells (RBCs) do not have capacity for gene expression, and instead possess an electrophysiological oscillator where cytosolic potassium plays a key role in timekeeping. We examined murine blood under normal conditions as well as in two perturbed states, malaria infection and induced anemia, to assess changes in baseline cellular electrophysiology and its implications for the electrophysiological oscillator. Blood samples were analyzed at 4-h intervals over 2 days by dielectrophoresis, and microscopic determination of parasitemia. We found that cytoplasmic conductivity (indicating the concentration of free ions in the cytoplasm and related to the membrane potential) exhibited circadian rhythmic behavior in all three cases (control, malaria and anemia). Compared to control samples, cytoplasm conductivity was decreased in the anemia group, whilst malaria-infected samples were in antiphase to control. Furthermore, we identified rhythmic behavior in membrane capacitance of malaria infected cells that was not replicated in the other samples. Finally, we reveal the historically famous rhythmicity of malaria parasite replication is in phase with cytoplasm conductivity. Our findings suggest the electrophysiological oscillator can impact on malaria parasite replication and/or is vulnerable to perturbation by rhythmic parasite activities.

Vm-related extracellular potentials observed in red blood cells.

Even in nonexcitable cells, the membrane potential Vm is fundamental to cell function, with roles from ion channel regulation, development, to cancer metastasis. Vm arises from transmembrane ion concentration gradients; standard models assume homogeneous extracellular and intracellular ion concentrations, and that Vm only exists across the cell membrane and has no significance beyond it. Using red blood cells, we show that this is incorrect, or at least incomplete; Vm is detectable beyond the cell surface, and modulating Vm produces quantifiable and consistent changes in extracellular potential. Evidence strongly suggests this is due to capacitive coupling between Vm and the electrical double layer, rather than molecular transporters. We show that modulating Vm changes the extracellular ion composition, mimicking the behaviour if voltage-gated ion channels in non-excitable channels. We also observed Vm-synchronised circadian rhythms in extracellular potential, with significant implications for cell-cell interactions and cardiovascular disease.

Dielectrophoresis as a Tool to Reveal the Potential Role of Ion Channels and Early Electrophysiological Changes in Osteoarthritis.

Diseases such as osteoarthritis (OA) are commonly characterized at the molecular scale by gene expression and subsequent protein production; likewise, the effects of pharmaceutical interventions are typically characterized by the effects of molecular interactions. However, these phenomena are usually preceded by numerous precursor steps, many of which involve significant ion influx or efflux. As a consequence, rapid assessment of cell electrophysiology could play a significant role in unravelling the mechanisms underlying drug interactions and progression of diseases, such as OA. In this study, we used dielectrophoresis (DEP), a technique that allows rapid, label-free determination of the dielectric parameters to assess the role of potassium ions on the dielectric characteristics of chondrocytes, and to investigate the electrophysiological differences between healthy chondrocytes and those from an in vitro arthritic disease model. Our results showed that DEP was able to detect a significant decrease in membrane conductance (6191 ± 738 vs. 8571 ± 1010 S/m2), membrane capacitance (10.3 ± 1.47 vs. 14.5 ± 0.01 mF/m2), and whole cell capacitance (5.4 ± 0.7 vs. 7.5 ± 0.3 pF) following inhibition of potassium channels using 10 mM tetraethyl ammonium, compared to untreated healthy chondrocytes. Moreover, cells from the OA model had a different response to DEP force in comparison to healthy cells; this was seen in terms of both a decreased membrane conductivity (782 S/m2 vs. 1139 S/m2) and a higher whole cell capacitance (9.58 ± 3.4 vs. 3.7 ± 1.3 pF). The results show that DEP offers a high throughput method, capable of detecting changes in membrane electrophysiological properties and differences between disease states.

Casein Kinase 1 Underlies Temperature Compensation of Circadian Rhythms in Human Red Blood Cells.

Temperature compensation and period determination by casein kinase 1 (CK1) are conserved features of eukaryotic circadian rhythms, whereas the clock gene transcription factors that facilitate daily gene expression rhythms differ between phylogenetic kingdoms. Human red blood cells (RBCs) exhibit temperature-compensated circadian rhythms, which, because RBCs lack nuclei, must occur in the absence of a circadian transcription-translation feedback loop. We tested whether period determination and temperature compensation are dependent on CKs in RBCs. As with nucleated cell types, broad-spectrum kinase inhibition with staurosporine lengthened the period of the RBC clock at 37°C, with more specific inhibition of CK1 and CK2 also eliciting robust changes in circadian period. Strikingly, inhibition of CK1 abolished temperature compensation and increased the Q10 for the period of oscillation in RBCs, similar to observations in nucleated cells. This indicates that CK1 activity is essential for circadian rhythms irrespective of the presence or absence of clock gene expression cycles.