Marco Stubbe

A short tutorial contribution to impedance and AC-electrokinetic characterization and manipulation of cells and media: Are electric methods more versatile than acoustic and laser methods?

Abstract Lab-on-chip systems (LOCs) can be used as in vitro systems for cell culture or manipulation in order to analyze or monitor physiological cell parameters. LOCs may combine microfluidic structures with integrated elements such as piezo-transducers, optical tweezers or electrodes for AC-electrokinetic cell and media manipulations. The wide frequency band (<1 kHz to >1 GHz) usable for AC-electrokinetic manipulation and characterization permits avoiding electrochemical electrode processes, undesired cell damage, and provides a choice between different polarization effects that permit a high electric contrast between the cells and the external medium as well as the differentiation between cellular subpopulations according to a variety of parameters. It has been shown that structural polarization effects do not only determine the impedance of cell suspensions and the force effects in AC-electrokinetics but can also be used for the manipulation of media with inhomogeneous temperature distributions. This manuscript considers the interrelations of the impedance of suspensions of cells and AC-electrokinetic single cell effects, such as electroorientation, electrodeformation, dielectrophoresis, electrorotation, and travelling wave (TW) dielectrophoresis. Unified models have allowed us to derive new characteristic equations for the impedance of a suspension of spherical cells, TW dielectrophoresis, and TW pumping. A critical review of the working principles of electro-osmotic, TW and electrothermal micropumps shows the superiority of the electrothermal pumps. Finally, examples are shown for LOC elements that can be produced as metallic structures on glass chips, which may form the bottom plate for self-sealing microfluidic systems. The structures can be used for cell characterization and manipulation but also to realize micropumps or sensors for pH, metabolites, cell-adhesion, etc.

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 Vrms. A further miniaturization of the pumps is viewed as quite feasible.

A new working principle for ac electro-hydrodynamic on-chip micro-pumps

Our new type of on-chip micro-pump exploits the ac electro-kinetic forces acting in the volume of a fluid in the presence of a temperature gradient. No mechanically movable parts are used. The velocity of the pump flow observed depends on the frequency and strength of the driving ac field and on the temperature gradient across the pump channel. An integrated heating element allows the temperature gradient to be adjusted. Both ac field electrodes and heating element are platinum structures processed on a glass chip. The pump-channel walls and cover are made from polymer and thin-glass, respectively. In this paper, we present measurements of the fluid velocity as functions of the medium conductivity (0.1–1.3 S m −1 ) and field frequency (300 kHz–52 MHz), voltage across the field-electrode voltage

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.

A decrease of intracellular ATP is compensated by increased respiration and acidification at sub-lethal parathion concentrations in murine embryonic neuronal cells: Measurements in metabolic cell-culture chips

We present a label-free in vitro method for testing the toxic potentials of chemical substances using primary neuronal cells. The cells were prepared from 16-day-old NMRI mouse embryos and cultured on silicon chips (www.bionas.de) under the influence of different parathion concentrations with sensors for respiration (Clark-type oxygen electrodes), acidification (pH-ISFETs) and cell adhesion (interdigitated electrode structures, IDES). After 12 days in vitro, the sensor readouts were simultaneously recorded for 350 min in the presence of parathion applying a serial 1:3 dilution. The parathion-dependent data was fitted by logistic functions. IC(50) values of approximately 105 μM, 65 μM, and 54 μM were found for respiration, acidification, and adhesion, respectively. An IC(50) value of approximately 36 μM was determined from the intracellular ATP-levels of cells, which were detected by an ATP-luminescence assay using micro-well plates. While the intracellular ATP level and cell adhesion showed no deviation from a simple logistic decay, increases of approximately 29% in the respiration and 15% in the acidification rates above the control values were found at low parathion concentrations, indicating hormesis. These increases could be fitted by a modified logistic function. We believe that the label-free, continuous, multi-parametric monitoring of cell-metabolic processes may have applications in systems-biology and biomedical research, as well as in environmental monitoring. The parallel characterization of IC(50) values and hormetic effects may provide new insights into the metabolic mechanisms of toxic challenges to the cell.

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 μm2. 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.

Impedance detection of the electrical resistivity of the wound tissue around deep brain stimulation electrodes permits registration of the encapsulation process in a rat model

Abstract An animal model of deep brain stimulation (DBS) was used in in vivo studies of the encapsulation process of custom-made platinum/iridium microelectrodes in the subthalamic nucleus of hemiparkinsonian rats via electrical impedance spectroscopy. Two electrode types with 100-μm bared tips were used: i) a unipolar electrode with a 200-μm diameter and a subcutaneous gold wire counter electrode and ii) a bipolar electrode with two parallelshifted 125-μm wires. Miniaturized current-controlled pulse generators (130 Hz, 200 μA, 60 μs) enabled chronic DBS of the freely moving animals. A phenomenological electrical model enabled recalculation of the resistivity of the wound tissue around the electrodes from daily in vivo recordings of the electrode impedance over two weeks. In contrast to the commonly used 1 kHz impedance, the resistivity is independent of frequency, electrode properties, and current density. It represents the ionic DC properties of the tissue. Significant resistivity changes were detected with a characteristic decrease at approximately the 2nd day after implantation. The maximum resistivity was reached before electrical stimulation was initiated on the 8th day, which resulted in a decrease in resistivity. Compared with the unipolar electrodes, the bipolar electrodes exhibited an increased sensitivity for the tissue resistivity.

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.

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.