Combined detection of AC-electrokinetic effects: Experiments with three-axial chicken red blood cells.

Dielectrophoresis (DEP), electrorotation (ROT), and electro-orientation were used for the dielectric spectroscopy of nucleated three-axial chicken red blood cells (CRBCs). Because the different AC-electrokinetic effects are not mutually independent, their DEP and ROT spectra were combined in ranges separated by the reorientation of the CRBCs in the inhomogeneous linear DEP and circular ROT fields. This behavior can be qualitatively described by a single-shell ellipsoidal model. Whereas in linear fields, the maximum of the Clausius-Mossotti factor along the three axes determines the orientated axis, in circular fields, the minimum of the factor determines the axis perpendicularly orientated to the field plane. Quantitatively, it has not been possible to find a consistent parameter set for fitting the DEP and ROT spectra, as well as the reorientation frequencies. Our ellipsoidal CRBC standard model had semiaxes of a = 7.7 µm, b = 4.0 µm, and c = 1.85 µm, a relative permittivity of 35 to 45 and conductivity of 0.36 to 0.04 S/m for the cytoplasm, combined with a specific capacitance of 10 to 14 mF/m2 and a conductivity of 3500 S/m2 for the cell membrane. The fits in different external conductivity ranges between external conductivities of 0.015 and 1.0 S/m were improved when the membrane capacitance was changed between 4 to 25 mF/m2 depending on the method used. A similar transition was reflected in the effective properties of a three-shell spherical model containing an internal membranous sphere with the geometry of the CRBC nucleus. Our findings suggest that the simultaneous interpretation of various AC-electrokinetic spectra is a step toward the dielectric fingerprinting of biological cells.

Furthering the state of knowledge on the electric properties of hemi-ellipsoidal single cells and cell patches on electrodes.

The impedance of electrodes with adherent biological cells correlates with cell viability and proliferation. To model this correlation, we exploited the idea that the introduction of a highly conductive layer into the equatorial equipotential slice of a system with an oriented, freely suspended, single ellipsoidal cell may split the system into mirror-symmetrical halves without changing the field distribution. Each half possesses half of the system's impedance and contains a hemiellipsoidal cell attached to the conductive layer, which can be considered a bottom electrode. For a hemiellipsoidal adherent cell model (ACM) with standard electrical properties for the external and cellular media, the assumption of a bottom membrane and a subcellular cleft in the 100 nm range, as found in adherent cells, changed the potential distribution over a one-% range up to frequencies of 1 MHz. For simplicity, potential distributions for slices of spheroidal objects can be numerically calculated in 2D. The 2D distributions can be converted into three dimensions using simplified equations for the influential radii of spheroids. After the ACM approach was expanded to adherent cell patch models (APMs), the feasibility of our model modifications was tested using two criteria: the constancy of the equipotential plane touching the poles of ACMs or APMs and a comparison of the impedance, which could be numerically calculated from the overall current between the bottom electrode and a plane-parallel counter-electrode, with the impedance of the suspension obtained from Maxwell-Wagner's mixing equation applied to hemiellipsoidal cells.

Neuronal in vitro activity is more sensitive to valproate than intracellular ATP: Considerations on conversion problems of IC50 in vitro data for animal replacement.

We investigated the effects of acute valproate (VPA) on mouse embryonic primary cortex cells (MEPCs). Intracellular ATP concentrations were compared with changes in the mean action potential (AP) frequencies of MEPC networks growing on microelectrode arrays. Our data implies biphasic reactions towards increasing VPA concentrations for both parameters. Intracellular ATP and mean AP frequencies increased around characteristic concentrations of 0.15 and 0.07mM to hormetic plateaus of approx. 120% and 160% of their controls, before fading around 17 and 1.7 mM, respectively. The biphasic in vitro behavior of the two parameters hinders a simple extraction of IC50 and Hillslope values. Different ways of data-fitting with single and double logistic functions are discussed. For a typical hormetic increase of 60% above control, IC50 and Hillslope were decreased by 37% and 15%, respectively. Despite these marginal effects at a logarithmic concentration scale, the hormetic and double logistic behavior of parameters may provide information on the mode of action of toxic compounds. Comparison of our values with the LD50 of mice, recalculated by normalization to body mass, suggests that a neurotoxic rather than a cytotoxic mechanism is killing the animals. The future use of cellular microsystems to replace animal experiments will motivate the development of new microsensors, as well as the consideration of newly accessible parameters in systems biology models.

Cell Monitoring and Manipulation Systems (CMMSs) based on Glass Cell-Culture Chips (GC³s).

We developed different types of glass cell-culture chips (GC³s) for culturing cells for microscopic observation in open media-containing troughs or in microfluidic structures. Platinum sensor and manipulation structures were used to monitor physiological parameters and to allocate and permeabilize cells. Electro-thermal micro pumps distributed chemical compounds in the microfluidic systems. The integrated temperature sensors showed a linear, Pt1000-like behavior. Cell adhesion and proliferation were monitored using interdigitated electrode structures (IDESs). The cell-doubling times of primary murine embryonic neuronal cells (PNCs) were determined based on the IDES capacitance-peak shifts. The electrical activity of PNC networks was detected using multi-electrode arrays (MEAs). During seeding, the cells were dielectrophoretically allocated to individual MEAs to improve network structures. MEA pads with diameters of 15, 20, 25, and 35 µm were tested. After 3 weeks, the magnitudes of the determined action potentials were highest for pads of 25 µm in diameter and did not differ when the inter-pad distances were 100 or 170 µm. Using 25-µm diameter circular oxygen electrodes, the signal currents in the cell-culture media were found to range from approximately -0.08 nA (0% O2) to -2.35 nA (21% O2). It was observed that 60-nm thick silicon nitride-sensor layers were stable potentiometric pH sensors under cell-culture conditions for periods of days. Their sensitivity between pH 5 and 9 was as high as 45 mV per pH step. We concluded that sensorized GC³s are potential animal replacement systems for purposes such as toxicity pre-screening. For example, the effect of mefloquine, a medication used to treat malaria, on the electrical activity of neuronal cells was determined in this study using a GC³ system.

Design and Characterization of a Sensorized Microfluidic Cell-Culture System with Electro-Thermal Micro-Pumps and Sensors for Cell Adhesion, Oxygen, and pH on a Glass Chip.

We combined a multi-sensor glass-chip with a microfluidic channel grid for the characterization of cellular behavior. The grid was imprinted in poly-dimethyl-siloxane. Mouse-embryonal/fetal calvaria fibroblasts (MC3T3-E1) were used as a model system. Thin-film platinum (Pt) sensors for respiration (amperometric oxygen electrode), acidification (potentiometric pH electrodes) and cell adhesion (interdigitated-electrodes structures, IDES) allowed us to monitor cell-physiological parameters as well as the cell-spreading behavior. Two on-chip electro-thermal micro-pumps (ETµPs) permitted the induction of medium flow in the system, e.g., for medium mixing and drug delivery. The glass-wafer technology ensured the microscopic observability of the on-chip cell culture. Connecting Pt structures were passivated by a 1.2 µm layer of silicon nitride (Si3N4). Thin Si3N4 layers (20 nm or 60 nm) were used as the sensitive material of the pH electrodes. These electrodes showed a linear behavior in the pH range from 4 to 9, with a sensitivity of up to 39 mV per pH step. The oxygen sensors were circular Pt electrodes with a sensor area of 78.5 µm(2). Their sensitivity was 100 pA per 1% oxygen increase in the range from 0% to 21% oxygen (air saturated). Two different IDES geometries with 30- and 50-µm finger spacings showed comparable sensitivities in detecting the proliferation rate of MC3T3 cells. These cells were cultured for 11 days in vitro to test the biocompatibility, microfluidics and electric sensors of our system under standard laboratory conditions.

Maxwell's mixing equation revisited: characteristic impedance equations for ellipsoidal cells.

We derived a series of, to our knowledge, new analytic expressions for the characteristic features of the impedance spectra of suspensions of homogeneous and single-shell spherical, spheroidal, and ellipsoidal objects, e.g., biological cells of the general ellipsoidal shape. In the derivation, we combined the Maxwell-Wagner mixing equation with our expression for the Clausius-Mossotti factor that had been originally derived to describe AC-electrokinetic effects such as dielectrophoresis, electrorotation, and electroorientation. The influential radius model was employed because it allows for a separation of the geometric and electric problems. For shelled objects, a special axial longitudinal element approach leads to a resistor-capacitor model, which can be used to simplify the mixing equation. Characteristic equations were derived for the plateau levels, peak heights, and characteristic frequencies of the impedance as well as the complex specific conductivities and permittivities of suspensions of axially and randomly oriented homogeneous and single-shell ellipsoidal objects. For membrane-covered spherical objects, most of the limiting cases are identical to-or improved with respect to-the known solutions given by researchers in the field. The characteristic equations were found to be quite precise (largest deviations typically <5% with respect to the full model) when tested with parameters relevant to biological cells. They can be used for the differentiation of orientation and the electric properties of cell suspensions or in the analysis of single cells in microfluidic systems.

The effect of hyperbaric air on the electric activity of neuronal in vitro networks.

Breathing hyperbaric air or gas mixtures, for example during diving or when working underwater is known to alter the electrophysiological behavior of neuronal cells, which may lead to restricted cognition. During the last few decades, only very few studies into hyperbaric effects have been published, especially for the most relevant pressure range of up to 10 bar. We designed a pressurized measuring chamber to record pressure effects on the electrical activity of neuronal networks formed by primary cells of the frontal cortex of NMRI mice. Electrical activity was recorded with multi-electrode arrays (MEAs) of glass neuro chips while subjected to a step-by-step pressure increase from atmospheric pressure (1 bar) to 2 and 4 bar, followed by a decompression to 1 bar, in order to record recovery effects. The effects of pressure on the total spike rates (TSRs), which were averaged from at least 45 chips, were detected in two cell culture media with different compositions. In a DMEM medium with 6% horse serum, the TSR was increased by 19% after a pressure increase to 2 bar and remained stable at 4 bar. In NMEM medium with 2% B27, the TSR was not altered by a pressure increase to 2 bar but increased by 9% at 4 bar. After decompression to 1 bar, the activities decreased to 76% and 101% of their respective control levels in the two media. MEA recordings from neuronal networks in miniaturized hyperbaric measuring chambers provide new access for exploring the neuronal effects of hyperbaric breathing gases.

Experimental verification of an equivalent circuit for the characterization of electrothermal micropumps: high pumping velocities induced by the external inductance at driving voltages below 5 V.

Electrothermal micropumps (ETµPs) use local heating to create conductivity and permittivity gradients in the pump medium. In the presence of such gradients, an external AC electric field influences smeared spatial charges in the bulk of the medium. When there is also a symmetry break, the field-charge interaction results in an effective volumetric force resulting in medium pumping. The advantages of the ETµP principle are the absence of moving parts, the opportunity to passivate all the pump structures, homogeneous pump-channel cross-sections, as well as force plateaus in broad frequency ranges. The ETµPs consisted of a DC-heating element and AC field electrodes arranged in a 1000 µm × 250 µm × 60 µm (length × width × height) channel. They were processed as platinum structures on glass carriers. An equivalent-circuit diagram allowed us to model the frequency-dependent pumping velocities of passivated and nonpassivated ETµPs, which were measured at medium conductivities up to 1.0 S/m in the 300 kHz to 52 MHz frequency range. The temperature distributions within the pumps were controlled by thermochromic beads. Under resonance conditions, an additional inductance induced a tenfold pump-velocity increase to more than 50 µm/s at driving voltages of 5 V(rms). A further miniaturization of the pumps is viewed as quite feasible.

Modular glass chip system measuring the electric activity and adhesion of neuronal cells--application and drug testing with sodium valproic acid.

We developed a modular neurochip system by combining a small (16x16 mm2) glass neurochip (GNC) with a homemade head stage and commercial data acquisition hardware and software. The system is designed for the detection of the electric activity of cultivated nerve or muscle cells by a 52-microelectrode array (MEA). In parallel, cell adhesion can be registered from the electric impedance of an interdigitated electrode structure (IDES). The GNC was tested with various cell lines and primary cells. It is fully autoclavable and re-useable. Murine embryonic primary cells were used as a model system to correlate the electric activity and adhesion of neuronal networks in a drug test with sodium valproic acid. The test showed the advantage of the parallel IDES and MEA measurements, i.e. the parallel detection of cytotoxic and neurotoxic effects. Toxic exposure of the cells during neuronal network formation allows for the characterization of developmental neurotoxic effects even at drug concentrations below the EC50-value for acute neurotoxic effects. At high drug concentrations, the degree of cytotoxic damage can still be assessed from the IDES data in the event that no electric activity develops. The GNC provides optimal cell culture conditions for up to months in combination with full microscopic observability. The 4'' glass wafer technology allows for a high precision of the GNC structures and an economic production of our new system that can be applied in general and developmental toxicity tests as well as in the search for neuro-active compounds.

Electrochemical product detection of an asymmetric convective polymerase chain reaction.

For the first time, we describe the application of heated microwires for an asymmetric convective polymerase chain reaction (PCR) in a modified PCR tube in a small volume. The partly single-stranded product was labeled with the electrochemically active compound osmium tetroxide bipyridine using a partially complementary protective strand with five mismatches compared to the single-stranded product. The labeled product could be successfully detected at a gold electrode modified with a complementary single-stranded capture probe immobilized via a thiol-linker. Our simple thermo-convective PCR yielded electrochemically detectable products after only 5-10 min. A significant discrimination between complementary and non-complementary target was possible using different immobilized capture probes. The total product yield was approx. half the amount of the classical thermocycler PCR. Numerical simulations describing the thermally driven convective PCR explain the received data. Discrimination between complementary capture probes and non-complementary capture probes was performed using square-wave voltammetry. The coupling of asymmetric thermo-convective PCR with electrochemical detection is very promising for future compact DNA sensor devices.

ac-Field-induced fluid pumping in microsystems with asymmetric temperature gradients.

We present two different designs of electrohydrodynamic micropumps for microfluidic systems. The micropumps have no movable parts, and their simple design allows for fabrication by microsystems technology. The pumps are operated by ac voltages from 1 to 60 V and were tested with aqueous solutions in the conductivity range of 1-112 mS m(-1). The pump effect is induced by an ac electric field across a fluid medium with an inhomogeneous temperature distribution. It is constant over a wide range of the ac field frequency with a conductivity-dependent drop-off at high frequencies. The temperature-dependent conductivity and permittivity distributions in the fluid induce space charges that interact with the electric field and induce fluid motion. The temperature distribution can be generated either by Joule heating in the medium or by external heating. We present experimental results obtained with two prototypes featuring Joule heating and external heating by a heating filament. Experimental and numerical results are compared with an analytical model.