Manipulation of microparticles for construction of array patterns by negative dielectrophoresis using multilayered array and grid electrodes

In this study, a useful method was developed to fabricate array patterns of microparticles not on electrode surfaces, but on arbitrary surfaces, using negative‐dielectrophoresis (n‐DEP). First, electrodes were designed and electric field simulations were performed to manipulate microparticles toward target areas. Based on the simulation results, multilayered array and grid (MLAG) electrodes, consisting of array electrodes surrounded by insulated regions and a grid electrode, were fabricated for the formation of localized, non‐uniform electric fields. The MLAG electrode was mounted to a target substrate in a face‐to‐face configuration with a spacer. When an AC voltage (4.60 V_rms and 1 MHz) was applied to the MLAG electrode, array patterns of 6 and 20 µm diameter microparticles were rapidly fabricated on the target substrate with ease. The results suggest that MLAG electrodes can be widely applied for the fabrication of biochips including cell arrays. Biotechnol. Bioeng. 2009; 104: 709–718 © 2009 Wiley Periodicals, Inc.     

Three dimensional electrode array for cell lysis via electroporation

Microfabricated devices for cell lysis have demonstrated many advantages over conventional approaches. Among various design of microdevices that employ electroporation for cytolysis, most utilize Ag/AgCl wires or 2D planar electrodes. Although, simple in fabrication the electric field generated by 2D electrodes decays exponentially, resulting in rather non-uniform forcing on the cell membrane. This paper investigates the effect of electric field generated by 3D cylindrical electrodes to perform cell lysis via electroporation in a microfluidic platform, and compared with that by 2D design. Computational results of the electric field for both 2D and 3D electrode geometries showed that the 3D configuration demonstrated a significantly higher effective volume ratio-volume which electric field is sufficient for cell lysis to that of net throughflow volume. Hence, the efficacy of performing cell lysis is substantially greater for cells passing through 3D than 2D electrodes. Experimentally, simultaneous multi-pores were observed on leukocytes lysed with 3D electrodes, which is indicative of enhanced uniformity of the electric field generated by 3D design. Additionally, a single row of 3D electrode demonstrated a substantially higher lysing percentage (30%) than that of 2D (8%) under that same flow condition. This work should aid in the design of electrodes in performing cell lysis via electroporation.     

Addition of magnetic field capability to existing extremely-low-frequency electric field exposure systems

Magnetic field systems were added to existing electric field exposure apparatuses for exposing cell suspensions in vitro and small animals in vivo. Two horizontally oriented, rectangular coils, stacked one directly above the other, have opposite electric currents. This configuration minimizes leakage fields and allows sham- and field-exposure systems to be placed in the same room or incubator. For the in vitro system, copper plates formed the loop-pair, with up to 900 A supplied by a 180:1 transformer. Electric fields were supplied via electrodes at the ends of cell-culture tubes, eight of which can be accommodated by each exposure system. Two complete systems are situated in an incubator to allow simultaneous sham and field exposure up to 1 mT. For the in vivo system, four pairs of 0.8 x 2.7-m coils made of copper bus bar are employed. This arrangement is energized from the power grid via a 30:1 transformer; horizontal magnetic flux densities up to 1 mT can be generated. Pairs of electrode plates spaced 30.5 cm apart provide electric field exposure of up to 130 kV/m. Four systems with a capacity of 48 rats each are located in one room. For both the in vitro and in vivo systems, magnetic exposure fields are uniform to within +/- 2.5%, and sham levels are at least 2,500-fold lower than exposure levels. Potential confounding factors, such as heating and vibration, were examined and found to be minimal.     

Influence of the current growth rate on the polarity effect in a wire array in the Angara-5-1 facility

It is shown that, at a sufficiently high current growth rate, the initial stage of implosion of a wire array is significantly affected by the radial electric fields. Due to the specific electrode configuration of wire arrays, the magnitude of the oppositely directed radial electric fields in different wire segments can reach 5 MV/cm. It is found that the process of plasma formation proceeds in different ways in segments with oppositely directed initial radial electric fields. The influence of this effect (the so-called “polarity effect”) on the implosion of cylindrical tungsten wire arrays in the Angara-5-1 facility becomes significant when the load voltage grows at a sufficiently high rate.     

Optimization of 2-D Electrode Configuration for Electrokinetic Remediation

A practical evaluation of one- and two-dimensional applications of electric fields for in situ extraction of contaminants is provided. The evaluation is based on contaminant transport by electroosmosis and ion migration. Parameters evaluated include electrode requirements, effectiveness of electric field distribution, remediation time, and energy expenditure. Formulation is provided for calculating cost components of the process, including electrode, energy, chemicals, posttreatment, fixed, and variable costs. Equations are also provided for evaluating optimum electrode spacings based on energy and time requirements. The derivations show that spacing between same-polarity electrodes is as significant in cost calculations and in process effectiveness as that between anodes and cathodes. Decreasing the same-polarity electrode spacing to half the anode-cathode spacing will result in a 100% increase in electrode requirements, but will decrease the area of the ineffective electric field by one half. Selection of the voltage gradient impacts the optimum electrode spacing. The analysis show that a minimum exists in the cost versus electrode spacings relationship.     

A separation chamber to sort cells and cell organelles by weak physical forces: III. Preparative electrophoresis of cells in stationary density gradients at low electric field strength

An apparatus was designed for preparative density gradient electrophoresis of mammalian cells. In a low conductivity isotonic Ficoll density gradient of 1.5 cm length, human erythrocytes treated with neuraminidase were separated from untreated erythrocytes at an electric field strength of approximately 2.7 v/cm. Within 5 min two bands of erythrocytes were visible. Electrophoretic separation was completed within 25 min. The fractionation is performed in a design consisting of three Perspex circular plates, bottom and top plates of which can be displaced simultaneously relative to the stationary middle plate by a worm-gear mechanism. The middle plate contains a cylindrical separation chamber of 50 cm² and 1.5 cm high. Top and bottom plates contain cones and flow deflectors for the undisturbed thin layering of cell suspensions and for introduction of the density gradient. Also present in top and bottom plates are electrode compartments containing a large platinum electrode and a cellophane membrane that isolates the separation chamber hydrodynamically but not electrically from the electrode compartment. The electrode compartments were flushed with electrophoresis buffer to remove products of electrophoresis as well as the (low) generated Joule heat.     

Quasi-Monopolar Stimulation: A Novel Electrode Design Configuration for Performance Optimization of a Retinal Neuroprosthesis

In retinal neuroprostheses, spatial interaction between electric fields from various electrodes – electric crosstalk – may occur in multielectrode arrays during simultaneous stimulation of the retina. Depending on the electrode design and placement, this crosstalk can either enhance or degrade the functional characteristics of a visual prosthesis. To optimize the device performance, a balance must be satisfied between the constructive interference of crosstalk on dynamic range and power consumption and its negative effect on artificial visual acuity. In the present computational modeling study, we have examined the trade-off in these positive and negative effects using a range of currently available electrode array configurations, compared to a recently proposed stimulation strategy – the quasi monopolar (QMP) configuration – in which the return current is shared between local bipolar guards and a distant monopolar electrode. We evaluate the performance of the QMP configuration with respect to the implantation site and electrode geometry parameters. Our simulation results demonstrate that the beneficial effects of QMP are only significant at electrode-to-cell distances greater than the electrode dimensions. Possessing a relatively lower activation threshold, QMP was found to be superior to the bipolar configuration in terms of providing a relatively higher visual acuity. However, the threshold for QMP was more sensitive to the topological location of the electrode in the array, which may need to be considered when programming the manner in which electrode are simultaneously activated. This drawback can be offset with a wider dynamic range and lower power consumption of QMP. Furthermore, the ratio of monopolar return current to total return can be used to adjust the functional performance of QMP for a given implantation site and electrode parameters. We conclude that the QMP configuration can be used to improve visual information-to-stimulation mapping in a visual prosthesis, while maintaining low power consumption.     

High density electromyography data of normally limbed and transradial amputee subjects for multifunction prosthetic control

Pattern recognition based control of powered upper limb myoelectric prostheses offers a means of extracting more information from the available muscles than conventional methods. By identifying repeatable patterns of muscle activity across multiple muscle sites rather than relying on independent EMG signals it is possible to provide more natural, reliable control of myoelectric prostheses. The purposes of this study were to (1) determine if participants can perform distinctive muscle activation patterns associated with multiple wrist and hand movements reliably and (2) to show that high density EMG can be applied individually to determine the electrode location of a clinically acceptable number of electrodes (maximally eight) to classify multiple wrist and hand movements reliably in transradial amputees. Eight normally limbed subjects (five female, three male) and four transradial amputee subjects (two traumatic and congenital) subjects participated in this study, which examined the classification accuracies of a pattern recognition control system. It was found that tasks could be classified with high accuracy (85-98%) with normally limbed subjects (10-13 tasks) and with amputees (4-6) tasks. In healthy subjects, reducing the number of electrodes to eight did not affect accuracy significantly when those electrodes were optimally placed, but did reduce accuracy significantly when those electrodes were distributed evenly. In the amputee subjects, reducing the number of electrodes up to 4 did not affect classification accuracy or the number of tasks with high accuracy, independent of whether those remaining electrodes were evenly distributed or optimally placed. The findings in healthy subjects suggest that high density EMG testing is a useful tool to identify optimal electrode sites for pattern recognition control, but its use in amputees still has to be proven. Instead of just identifying the electrode sites where EMG activity is strong, clinicians will be able to choose the electrode sites that provide the most important information for classification.     

Single-cell electroporation

Electroporation is a widely used method for the introduction of polar and charged agents such as dyes, drugs, DNA, RNA, proteins, peptides, and amino acids into cells. Traditionally, electroporation is performed with large electrodes in a batch mode for treatment of a large number of cells in suspension. Recently, microelectrodes that can produce extremely localized electric fields, such as solid carbon fiber microelectrodes, electrolyte-filled capillaries and micropipettes as well as chip-based microfabricated electrode arrays, have proven useful to electroporate single cells and subcellular structures. Single-cell electroporation opens up a new window of opportunities in manipulating the genetic, metabolic, and synthetic contents of single targeted cells in tissue slices, cell cultures, in microfluidic channels or at specific loci on a chip-based device.     

From Oscillatory Transcranial Current Stimulation to Scalp EEG Changes: A Biophysical and Physiological Modeling Study

Both biophysical and neurophysiological aspects need to be considered to assess the impact of electric fields induced by transcranial current stimulation (tCS) on the cerebral cortex and the subsequent effects occurring on scalp EEG. The objective of this work was to elaborate a global model allowing for the simulation of scalp EEG signals under tCS. In our integrated modeling approach, realistic meshes of the head tissues and of the stimulation electrodes were first built to map the generated electric field distribution on the cortical surface. Secondly, source activities at various cortical macro-regions were generated by means of a computational model of neuronal populations. The model parameters were adjusted so that populations generated an oscillating activity around 10 Hz resembling typical EEG alpha activity. In order to account for tCS effects and following current biophysical models, the calculated component of the electric field normal to the cortex was used to locally influence the activity of neuronal populations. Lastly, EEG under both spontaneous and tACS-stimulated (transcranial sinunoidal tCS from 4 to 16 Hz) brain activity was simulated at the level of scalp electrodes by solving the forward problem in the aforementioned realistic head model. Under the 10 Hz-tACS condition, a significant increase in alpha power occurred in simulated scalp EEG signals as compared to the no-stimulation condition. This increase involved most channels bilaterally, was more pronounced on posterior electrodes and was only significant for tACS frequencies from 8 to 12 Hz. The immediate effects of tACS in the model agreed with the post-tACS results previously reported in real subjects. Moreover, additional information was also brought by the model at other electrode positions or stimulation frequency. This suggests that our modeling approach can be used to compare, interpret and predict changes occurring on EEG with respect to parameters used in specific stimulation configurations.     

Uniform magnetic fields and double-wrapped coil systems: improved techniques for the design of bioelectromagnetic experiments.

A common mistake in biomagnetic experimentation is the assumption that Helmholtz coils provide uniform magnetic fields; this is true only for a limited volume at their center. Substantial improvements on this design have been made during the past 140 years with systems of three, four, and five coils. Numerical comparisons of the field uniformity generated by these designs are made here, along with a table of construction details and recommendations for their use in experiments in which large volumes of uniform intensity magnetic exposures are needed. Double-wrapping, or systems of bifilar windings, can also help control for the non-magnetic effects of the electric coils used in many experiments. In this design, each coil is wrapped in parallel with two separate, adjacent strands of copper wire, rather than the single strand used normally. If currents are flowing in antiparallel directions, the magnetic fields generated by each strand will cancel and yield virtually no external magnetic field, whereas parallel currents will yield an external field. Both cases will produce similar non-magnetic effects of ohmic heating, and simple measures can reduce the small vibration and electric field differences. Control experiments can then be designed such that the only major difference between treated and untreated groups is the presence or absence of the magnetic field. Double-wrapped coils also facilitate the use of truly double-blind protocol, as the same apparatus can be used either for experimental or control groups.     

Reduction of electrode polarization capacitance in low-frequency impedance spectroscopy by using mesh electrodes

Dielectric measurements of biological samples are obscured by electrode polarization, which at low frequencies dominates over the actual sample response. Reduction of this artifact is especially necessary in studying interactions of electric field with biological systems in the α-dispersion range. We developed a method to reduce the influence of electrode polarization by employing mesh instead of solid electrodes as sensing probes, thereby reducing the area of the double layer. The design decreases the electrode-electrolyte contact area by almost 40% while keeping the bulk sample capacitance the same. Interrogation electric fields away from the electrode surface and sensitivity are unaffected. Electrodes were microfabricated (600μm×50μm, spacing of 100μm) with and without mesh holes 7.5μm×7.5μm in size. Simulations of electric field performed using Comsol Multiphysics showed non-uniformity of the electric field within less than 1.5μm from the electrode surface, which encompasses the double layer region, but at greater distance the solid and mesh electrodes gave the same results. Mesh electrodes reduced capacitance measurements for water and KCl solutions of different concentrations at low frequencies (<10kHz), while higher frequency capacitance remained the same for both electrode types, confirming our hypothesis that this design leaves the electric field mainly unaffected. Impedance measurements at low frequencies for water and mice heart mitochondrial suspension were lower for mesh than for solid electrodes. Comsol simulations confirmed these results by showing that mesh electrodes have a greater charge density than solid electrodes, which affects conductance. These electrodes are being used for mitochondrial membrane potential studies.     

Single cell and neural process experimentation using laterally applied electrical fields between pairs of closely apposed microelectrodes with vertical sidewalls

As biomedical research has moved increasingly towards experimentation on single cells and subcellular structures, there has been a need for microscale devices that can perform manipulation and stimulation at a correspondingly small scale. We propose a microelectrode array (MEA) featuring thickened microelectrodes with vertical sidewalls (VSW) to focus electrical fields horizontally on targets positioned in between paired electrodes. These microelectrodes were fabricated using gold electroplating that was molded by photolithographically patterned SU-8 photoresist. Finite element modeling showed that paired VSW electrodes produce more uniform electrical fields compared to conventional planar microelectrodes. Using paired microelectrodes, 3 μm thick and spaced 10 μm apart, we were able to perform local electroporation of individual axonal processes, as demonstrated by entry of EGTA to locally chelate intra-axonal calcium, quenching the fluorescence of a pre-loaded calcium indicator dye. The same electrode configuration was used to electroporate individual cells, resulting in the targeted transfection of a transgene expressing a cytoplasmically soluble green fluorescent protein (GFP). In addition to electropration, our electrode configuration was also capable of precisely targeted field stimulation on individual neurons, resulting in action potentials that could be tracked by optical means. With its ability to deliver well-characterized electrical fields and its versatility, our configuration of paired VSW electrodes may provide the basis for a new tool for high-throughput and high-content experimentation in broad areas of neuroscience and biomedical research.     

Electric field-assisted biosorption

A bisorption process using electric fields to facilitate contact between a sorbate and non-living biomass is described. The latter is enclosed within a semi-permeable membrane together with an electrode. The counter electrode is placed in the sorbate solution and an established potential across the electrodes facilitates electrokinetic movement of the sorbate to the biosorbant material.     

Multiphase electrodes for microbead control applications: integration of DEP and electrokinetics for bio-particle positioning

Advances in microfabrication have introduced new possibilities for automated, high-throughput biomedical investigations and analysis. Physical effects such as dielectrophoresis (DEP) and AC electrokinetics can be used to manipulate particles in solution to coordinate a sequence of bioanalytical processing steps. DEP is accomplished with non-uniform electric fields that can polarize particles (microbeads, cells, viruses, DNA, proteins, etc.) in suspension causing translational or rotational movement. AC electrokinetics is another phenomena involved with movement of particles in suspension with electric fields and is comprised of both electro-thermal and electro-osmotic effects. This paper investigates single layer electrodes that are effective for particle localization and clustering based on DEP and AC electrokinetic effects. We demonstrate a novel multi-electrode setup capable of clustering particles into an array of discrete bands using activated and electrically floating electrodes. These bands shift to adjacent regions on the electrode surface by altering the electrode activation scheme. The predictability of particle placement to specific locations provides new opportunities for integration and coordination with raster scanning lasers or a charge coupled device (CCD) for advanced biomedical diagnostic devices, and more sophisticated optical interrogation techniques.     

Spatial structure of emission from an electrode microwave discharge in hydrogen

The structure of electrode microwave (2.45 GHz) discharges in hydrogen with electrodes of various shapes and sizes at pressures of 1–8 torr and incident powers of 2–150 W is studied. It is found that the discharges exhibit a common feature that is independent of the antenna-electrode design: near the electrode surface, there is a thin bright sheath surrounded by a less bright, sharply bounded region, which is usually shaped like a sphere. It is suggested that the structure observed arises because the microwave field maintaining the discharge is strongly nonuniform. Near the electrode, there exists a thin dense plasma sheath with a high electron density gradient. A strong dependence of the electron-impact excitation coefficient on the electric field makes the effect even more pronounced. As the electron density decreases due to dissociative recombination, the microwave field gradient decreases and the discharge emission intensity tends to a nearly constant value. Presumably, in the boundary region of the discharge, there exists a surface wave, which increases the emission intensity at the periphery of the discharge.     

Brain stimulation using electromagnetic sources: theoretical aspects.

We prove that, at the frequencies generally proposed for extracranial stimulation of the brain, it is not possible, using any superposition of external current sources, to produce a three-dimensional local maximum of the electric field strength inside the brain. The maximum always occurs on a boundary where the conductivity jumps in value. Nevertheless, it may be possible to achieve greater two-dimensional focusing and shaping of the electric field than is currently available. Towards this goal we have used the reciprocity theorem to present a uniform treatment of the electric field inside a conducting medium produced by a variety of sources: an external magnetic dipole (current loop), an external electric dipole (linear antenna), and surface and depth electrodes. This formulation makes use of the lead fields from magneto- and electroencephalography. For the special case of a system with spherically symmetric conductivity, we derive a simple analytic formula for the electric field due to an external magnetic dipole. This formula is independent of the conductivity profile and therefore embraces spherical models with any number of shells. This explains the "insensitivity" to the skull's conductivity that has been described in numerical studies. We also present analytic formulas for the electric field due to an electric dipole, and also surface and depth electrodes, for the case of a sphere of constant conductivity.     

Construction of a very high-density extracellular electrode array

Cellular activation mapping (specifying in time and space the electrical activation sequence of cells) is a well-established basic research tool in cardiac, neural, and gastric physiology. Much recent research in cardiac mapping has focused on large arrays (>200 electrodes) with small electrodes (<500 microm). Construction of such arrays using standard techniques is tedious and yields irregular electrode spacing. We present a novel construction technique that rapidly produces large arrays with regularly spaced small electrodes. For methods, fine-pitch copper ribbon cables, insulated with either polyvinylchloride (PVC) or polyimide (flexible printed circuit; FPC), were assembled together such that the active surface was the cut end of the cable. The cut end was sanded and polished, then coated with silver and sometimes silver chloride. Once completed, the alternating current (AC) root-mean-square (rms) potential was measured between two adjacent, individual electrodes. Polarization testing was conducted according to a previously reported protocol (Witkowski FX and Penkoske PA. J Electrocardiol 21: 273-282, 1988). Activation mapping was conducted in the open-chest guinea pig with both pacing- and defibrillation- strength stimuli. In terms of results, four PVC and three FPC arrays were constructed, ranging from 4 to 400 electrodes. Two hours of labor were needed to create a complete electrode array, independent of the number of electrodes, including connectors and silver/silver chloride coating. As expected, the addition of a silver/silver chloride coating significantly reduced (0.76-0.42 mV, P < 0.001) the AC rms potential difference between two electrodes. A nearly immediate recovery of the potential difference between adjacent pairs of silver/silver chloride electrodes was observed after defibrillation stimuli.     

In vivo 3-D distributions of electric fields in pig skin with rectangular pulse electrical current stimulation (RPECS)

We developed stimulating and detecting electrodes. We experimentally examined three dimensional (3-D) distributions of electric fields in living pig skin under and around the stimulating electrodes with the detecting electrodes and rectangular pulsed electrical current stimulation (RPECS). We verified our previous physical assumption, E ≈ I / (A σ_dz), in the skin under the electrode, where E, I, A and σ_dz respectively represent the electric field, the externally imposed peak current, the cross sectional area of the stimulating electrode and the perpendicular conductivity of the skin. Pulses were 30 mA, 140 μs and 128 pulses per second (pps). These parameters were previously used in our laboratory to enhance cutaneous regeneration, in vivo, with RPECS. © 1996 Wiley-Liss, Inc.     

Investigation of the temporal and spatial dynamics of muscular action potentials through optically pumped magnetometers

AimThis study aims to simultaneously record the magnetic and electric components of the propagating muscular action potential.MethodA single-subject study of the monosynaptic stretch reflex of the musculus rectus femoris was performed; the magnetic field generated by the muscular activity was recorded in all three spatial directions by five optically pumped magnetometers. In addition, the electric field was recorded by four invasive fine-wire needle electrodes. The magnetic and electric fields were compared by modelling the muscular anatomy of the rectus femoris muscle and by simulating the corresponding magnetic field vectors.ResultsThe magnetomyography (MMG) signal can reliably be recorded following the stimulation of the monosynaptic stretch reflex. The MMG signal shows several phases of activity inside the muscle, the first of which is the propagating muscular action potential. As predicted by the finite wire model, the magnetic field vectors of the propagating muscular action potential are generated by the current flowing along the muscle fiber. Based on the magnetic field vectors, it was possible to reconstruct the pinnation angle of the muscle fibers. The later magnetic field components are linked to the activation of the contractile apparatus.InterpretationMMG allows to analyze the muscle physiology from the propagating muscular action potential to the initiation of the contractile apparatus. At the same time, this methods reveals information about muscle fiber direction and extend. With the development of high-resolution magnetic cameras, that are based on OPM technology, it will be possible to image the function and structure of the biomagnetic field of any skeletal muscle with high precision. This method could be used both, in clinical medicine and also in sports science.