Low Voltage Pulse Research Articles

Electrotransfer parameters as a tool for controlled and targeted gene expression in skin

Skin is an attractive target for gene electrotransfer. It consists of different cell types that can be transfected, leading to various responses to gene electrotransfer. We demonstrate that these responses could be controlled by selecting the appropriate electrotransfer parameters. Specifically, the application of low or high electric pulses, applied by multi-electrode array, provided the possibility to control the depth of the transfection in the skin, the duration and the level of gene expression, as well as the local or systemic distribution of the transgene. The influence of electric pulse type was first studied using a plasmid encoding a reporter gene (DsRed). Then, plasmids encoding therapeutic genes (IL-12, shRNA against endoglin, shRNA against melanoma cell adhesion molecule) were used, and their effects on wound healing and cutaneous B16F10 melanoma tumors were investigated. The high-voltage pulses resulted in gene expression that was restricted to superficial skin layers and induced a local response. In contrast, the low-voltage electric pulses promoted transfection into the deeper skin layers, resulting in prolonged gene expression and higher transgene production, possibly with systemic distribution. Therefore, in the translation into the clinics, it will be of the utmost importance to adjust the electrotransfer parameters for different therapeutic approaches and specific mode of action of the therapeutic gene.

An Efficient High Voltage Level Shifter using Coupling Capacitor for a High Side Buck Converter

We propose an efficient high voltage level shifter for a high side Buck converter driving a light-emitting diode (LED) lamp. The proposed circuit is comprised of a low voltage pulse width modulation (PWM) signal driver, a coupling capacitor, a resistor, and a diode. The proposed method uses a property of a PWM signal. The property is that the signal repeatedly transits between a low and high level at a certain frequency. A low voltage PWM signal is boosted to a high voltage PWM signal through a coupling capacitor using the property of the PWM signal, and the boosted high voltage PWM signal drives a p-channel metal oxide semiconductor (PMOS) transistor on the high side Buck converter. Experimental results show that the proposed level shifter boosts a low voltage (0 to 20 V) PWM signal at 125 kHz to a high voltage (370 to 380 V) PWM signal with a duty ratio of up to 0.9941.

Design of series-connected thyristor switch and synchronous triggering circuit

In order to meet the requirement of pulsed power source on high voltage and large current switches，a series-connected thyristor switch with a rated voltage of 10 kV and a rated current of 500 A was developed the traditional voltage sharing method. According to the principle of thyristor trigger, a trigger system was developed for synchronous triggering multiple thyristors. As to generate low-voltage pulse with variable pulse widths, the trigger system used an IGBT switch to cut the DC voltage. In the trigger system, the voltage amplitude of the pulse was increased and the synchronous trigger signals were generated through some isolated pulse transformers. Finally, the thyristor trigger system and the series-connected thyristor switch were tested, Showing that the trigger system could produce multi-channel synchronous trigger signals with a voltage of 20 V, a current of 1 A, a pulse repetition rate of 100 Hz and pulse durations from 30 s to 60 s. Static voltage fluctuation and active voltage fluctuation of the series-connected thyristor switch were small, indicating it could reliably work under the condition of high voltage and large current.

Electrochemical micromachining of small tapered microstructures with sub-micro spherical tool

A sub-micro spherical tool electrode is introduced for electrochemical micromachining (EMM), which could reduce the taper angle of the processed microstructures. An experimental setup that includes macro stages and piezo nano-positioning actuators is developed for EMM. The sub-micro spherical tool is prepared by liquid membrane pulsed electrochemical machining. The stability of the EMM process is in-process monitored by acquiring both the voltage and current signals. The machining depth achieved with the sub-micro spherical tool is 139 % of that achieved with a sharp conical tool. It is also indicated that the machining stability could also be improved with the spherical tool. Experimental results reveal that a low pulse voltage level, small pulse duration, and low electrolyte concentration decrease the machining side gap. Small tapered microstructures such as micro-cube of 30 × 30 × 0.35 μm in size and microfluidic channels with varying widths are manufactured by EMM.

Microstructure of a-C:H films prepared on a microtrench and analysis of ions and radicals behavior

Amorphous carbon films (a-C:H) were prepared on a microtrench (4-μm pitch and 4-μm depth), and the uniformity of film thickness and microstructure of the films on the top, sidewall, and bottom surfaces of the microtrench were evaluated by scanning electron microscopy and Raman spectroscopy. The a-C:H films were prepared by bipolar-type plasma based ion implantation and deposition (bipolar PBII&D), and the negative pulse voltage, which is the main parameter dominating the film structure, was changed from −1.0 to −15 kV. Moreover, the behavior of ions and radicals was analyzed simultaneously by combining the calculation methods of Particle-In-Cell/Monte Carlo Collision (PIC-MCC) and Direct Simulation Monte Carlo (DSMC) to investigate the coating mechanism for the microtrench. The results reveal that the thickness uniformity of a-C:H films improves with decreasing negative pulse voltage due to the decreasing inertia of incoming ions from the trench mouth, although the film thickness on the sidewall tends to be much smaller than that on the top and bottom surfaces of the trench. The normalized flux and the film thickness show similar behavior, i.e., the normalized flux or thickness at the bottom surface increases at low negative pulse voltages and then saturates at a certain value, whereas at the sidewall it monotonically decreases with increasing negative voltage. The microstructure of a-C:H films on the sidewall surface is very different from that on the top and bottom surfaces. The film structure at a low negative pulse voltage shifts to more of a polymer-like carbon (PLC) structure due to the lower incident energy of ions. Although the radical flux on the sidewall increases slightly, the overall film structure is not significantly changed because this film formation at a low negative voltage is originally dominated by radicals. On the other hand, the flux of radicals is dominant on the sidewall in the case of high negative pulse voltage, resulting in a deviation from the Raman behavior of a-C:H films deposited by bipolar PBII&D. This tendency intensifies as the negative voltage becomes greater. Also, the energy of incident ions on the sidewall of the trench increases with increasing negative voltage, which causes a shift in the Raman data of the sidewall to the bottom right corner on the figure depicting the relationship of the FWHM(G) and the G-peak position, indicating increased graphitization of a-C:H film.

Wave trains induced by circularly polarized electric fields in cardiac tissues.

Clinically, cardiac fibrillation caused by spiral and turbulent waves can be terminated by globally resetting electric activity in cardiac tissues with a single high-voltage electric shock, but it is usually associated with severe side effects. Presently, a promising alternative uses wave emission from heterogeneities induced by a sequence of low-voltage uniform electric field pulses. Nevertheless, this method can only emit waves locally near obstacles in turbulent waves and thereby requires multiple obstacles to globally synchronize myocardium and thus to terminate fibrillation. Here we propose a new approach using wave emission from heterogeneities induced by a low-voltage circularly polarized electric field (i.e., a rotating uniform electric field). We find that, this approach can generate circular wave trains near obstacles and they propagate outwardly. We study the characteristics of such circular wave trains and further find that, the higher-frequency circular wave trains can effectively suppress spiral turbulence.

Tree initiation characteristics of epoxy resin in Ln<sub>2</sub> for superconducting magnet insulation

The superconducting magnet system is the key part of international thermonuclear experimental reactor (ITER) device which ensures the feasibility of fusion energy application. Epoxy resin has been used as the adhesives and electrical insulation in the magnet system. Since ITER device operates in the liquid nitrogen environment and is driven by a pulse power, the insulation made of epoxy resin faces the challenges of extremely low temperature and pulse voltage. This paper investigated the influence of low temperature on the electrical tree characteristics in epoxy resin. Positive pulse voltage with the frequency of 400 Hz was applied on the needle-plate geometry electrodes. The amplitudes of the pulse voltage were 10, 12 and 14 kV. In order to simulate the practical defects, a metal needle was inserted into the samples and the distance between the tip and plane was 2 mm. The experimental temperature ranged from 30 to -196 °C. The experimental results indicate that the low temperature have a significant influence on the electrical tree characteristics. Different tree structures in epoxy resin are observed with the changing temperatures. The density of the tree branches increases as the temperature decreases, while the tree growth rate presents an opposite trend. It is also revealed that the time to breakdown become long under the low temperature. However, once the breakdown occurs, a wide breakdown channels will be left in epoxy resin, which means great destruction to the electrical insulation. In addition, when the samples are under the same ambient temperature, the probability of tree initiation shows a significant increase and electrical trees grow faster in the following period with application of higher pulse voltage. The higher-energy charges may be responsible for the high growth rate.

Reducing error rates in straintronic multiferroic nanomagnetic logic by pulse shaping

Dipole-coupled nanomagnetic logic (NML), where nanomagnets (NMs) with bistable magnetization states act as binary switches and information is transferred between them via dipole-coupling and Bennett clocking, is a potential replacement for conventional transistor logic since magnets dissipate less energy than transistors when they switch in a logic circuit. Magnets are also ‘non-volatile’ and hence can store the results of a computation after the computation is over, thereby doubling as both logic and memory—a feat that transistors cannot achieve. However, dipole-coupled NML is much more error-prone than transistor logic at room temperature because thermal noise can easily disrupt magnetization dynamics. Here, we study a particularly energy-efficient version of dipole-coupled NML known as straintronic multiferroic logic (SML) where magnets are clocked/switched with electrically generated mechanical strain. By appropriately ‘shaping’ the voltage pulse that generates strain, we show that the error rate in SML can be reduced to tolerable limits. We describe the error probabilities associated with various stress pulse shapes and discuss the trade-off between error rate and switching speed in SML.The lowest error probability is obtained when a ‘shaped’ high voltage pulse is applied to strain the output NM followed by a low voltage pulse. The high voltage pulse quickly rotates the output magnet’s magnetization by 90° and aligns it roughly along the minor (or hard) axis of the NM. Next, the low voltage pulse produces the critical strain to overcome the shape anisotropy energy barrier in the NM and produce a monostable potential energy profile in the presence of dipole coupling from the neighboring NM. The magnetization of the output NM then migrates to the global energy minimum in this monostable profile and completes a 180° rotation (magnetization flip) with high likelihood.

Transport, resealing, and re-poration dynamics of two-pulse electroporation-mediated molecular delivery

Electroporation is of interest for many drug-delivery and gene-therapy applications. Prior studies have shown that a two-pulse-electroporation protocol consisting of a short-duration, high-voltage first pulse followed by a longer, low-voltage second pulse can increase delivery efficiency and preserve viability. In this work the effects of the field strength of the first and second pulses and the inter-pulse delay time on the delivery of two different-sized Fluorescein–Dextran (FD) conjugates are investigated. A series of two-pulse-electroporation experiments were performed on 3T3-mouse fibroblast cells, with an alternating-current first pulse to permeabilize the cell, followed by a direct-current second pulse. The protocols were rationally designed to best separate the mechanisms of permeabilization and electrophoretic transport. The results showed that the delivery of FD varied strongly with the strength of the first pulse and the size of the target molecule. The delivered FD concentration also decreased linearly with the logarithm of the inter-pulse delay. The data indicate that membrane resealing after electropermeabilization occurs rapidly, but that a non-negligible fraction of the pores can be reopened by the second pulse for delay times on the order of hundreds of seconds. The role of the second pulse is hypothesized to be more than just electrophoresis, with a minimum threshold field strength required to reopen nano-sized pores or defects remaining from the first pulse. These results suggest that membrane electroporation, sealing, and re-poration is a complex process that has both short-term and long-term components, which may in part explain the wide variation in membrane-resealing times reported in the literature.

Phase Transition Experimental and Theoretical Study of Micro Power Generator Supplying Source for CMOS Chip Based on Ferroelectric Ceramic Nano-Porous Material.

We demonstrated both experimentally and in theory analysis and calculation that the tin-modified lead zirconate titanate nanoporous ferroelectric generator system can perform as a micro-power supplying source for CMOS chip. The ferroelectric ceramic phase transition under transverse shock wave compression can charge external storage capacitor. The nanoporous microstructure ferro-electric ceramic micro-pulsed-power system is capable of generating low output voltage pulses and supplying CMOS chip with micro power sources. We developed the methodology for theory analysis and experimental operation of the ferroelectric generator. Analysis of the porous ferroelectric ceramic material was carried out by X-ray diffractometry and X-ray photoelectron spectroscopy. Microstructures and surface morphology of porous ferroelectric ceramics samples were examined by using scanning electron microscopy. The planar shock wave experiments were conducted on a compressed-gas gun. The experimental results were in good agreement with the theory analysis. Keywords: PSZT Ferroelectric Ceramic, Shock Wave, Phase Transition, Depolarization, Micro-Power-Generator.

New insights into the mechanisms of gene electrotransfer--experimental and theoretical analysis.

Gene electrotransfer is a promising non-viral method of gene delivery. In our in vitro study we addressed open questions about this multistep process: how electropermeabilization is related to electrotransfer efficiency; the role of DNA electrophoresis for contact and transfer across the membrane; visualization and theoretical analysis of DNA-membrane interaction and its relation to final transfection efficiency; and the differences between plated and suspended cells. Combinations of high-voltage and low-voltage pulses were used. We obtained that electrophoresis is required for the insertion of DNA into the permeabilized membrane. The inserted DNA is slowly transferred into the cytosol, and nuclear entry is a limiting factor for optimal transfection. The quantification and theoretical analysis of the crucial parameters reveals that DNA-membrane interaction (NDNA) increases with higher DNA concentration or with the addition of electrophoretic LV pulses while transfection efficiency reaches saturation. We explain the differences between the transfection of cell suspensions and plated cells due to the more homogeneous size, shape and movement of suspended cells. Our results suggest that DNA is either translocated through the stable electropores or enters by electo-stimulated endocytosis, possibly dependent on pulse parameters. Understanding of the mechanisms enables the selection of optimal electric protocols for specific applications.

The dependence of efficiency of transmembrane molecular transfer using electroporation on medium viscosity.

In the present study, we aimed to evaluate the efficiency of drug and gene electrotransfer into cells in vitro depending on medium viscosity. Experiments were performed using Chinese hamster ovary cells. Efficiency of molecular electrotransfer depending of medium viscosity was evaluated using two different electroporation conditions: a high-voltage (HV) pulse and a combination of a high-voltage pulse and a low-voltage pulse (HV + LV). To evaluate the efficiency of molecular electrotransfer, anticancer drug bleomycin and two different plasmids coding for green fluorescent protein and luciferase were used. We found that a slight increase in medium viscosity from 1.3-1.4 mPa·s significantly decreased the transfection efficiency, both in terms of transfected cells and total protein production, which was abolished completely with an increase in medium viscosity to 6.1 mPa·s. Notably, at this medium viscosity, electrotransfer of the small anticancer drug was still efficient. Using HV and HV + LV pulse combinations, we showed that a decrease of DNA electrotransfer, especially at lower medium viscosities, can be compensated for by the LV pulse to some extent. On the other hand, the addition of the LV pulse after the HV pulse did not have any positive effect on the efficiency of bleomycin electrotransfer. These findings demonstrate that transfection is very susceptible to medium viscosity and highlights the importance of the electrophoretic component in experiments when a considerable transfection level is needed.

Investigation of the characteristics of DLC films on oxynitriding-treated ASP23 high speed steel by DC-pulsed PECVD process

In this study, diamond-like carbon (DLC) films are coated onto oxynitriding-treated ASP23 high-speed steel by DC-pulsed PECVD. The main research parameters of the DC-pulsed PECVD process are the various duty cycles (5%, 10%, 15% and 20%) used to examine the coating characteristics. In order to investigate the DLC film properties, a Raman spectroscopy analysis, wear test, adhesion, and hardness tests are performed. The experimental results show that the duplex coating layers have optimal properties when DLC films are treated with a low-pulse voltage (−1.5kV), a coating time of 90min and duty cycles maintained at 10%. As a result, the DLC/oxynitriding duplex-treated specimens with the highest surface hardness (Hv0.01 1931) and the lowest wear volume loss (3×10−3mm3) can be acquired. In addition, compared with non-treated (polarization resistance Rp=5×103Ωcm2) and oxynitriding-treated (Rp=8×103Ωcm2) specimens, the optimal DLC/oxynitriding duplex-treated ASP23 high-speed steel possesses the lowest corrosion current (Icorr=8×10−6Acm−2) and highest polarization resistance (Rp=1×104Ωcm2) in 3.5wt.% NaCl solutions. This result confirms that the DLC/oxynitriding duplex treatment enhances optimal wear and corrosion resistance.

Design and Performance of Microdevices for High-Efficiency Sterilization of Bacteria

Sterilization of bacteria is a important aspect in food safety, health, and experimental study. There are a lot of sterilization methods, which are mainly divided into two categories: heating and non-heating. Pulsed electric field (PEF) based sterilization has become a widely used non-heating method due to its outstanding advantages such as fast, low energy consumption and temperature, high efficiency, no chemical byproduct, little change of the nutrition of liquid foods, and so on. However, high-voltage generator is required in the traditional PEF sterilization device, and it is too costly and dangerous to be widely applied.In this study, devices with micro-scale electrodes are designed for the sterilization of bacteria. Due to much short distance between each pair of electrodes, low voltage pulse electric field can be used to achieve good effect of sterilization. Structures of electrodes and channels are attentively designed for desired electric-field profile, the impact of some electric parameters is also discussed.COMSOL Multiphysics software is used to simulate the change of trans-membrane voltage on bacteria and the electroporation process. Geometries of electrodes and channels, as well as some electric parameters such as voltage, number, width, interval of electric pulses are investigated. Simulation results show that the electric field can be focused by microelectrode array other than plate electrodes with micro-scale gap. Thus, stronger electric field can be generated within a microelectrode array than that between two plate electrodes under the same applied voltage, and it helps sterilization. For three different microelectrode arrays, planar-comb teeth structure can achieve better sterilization effect. The impact of electric parameters on the generation, amplitude and distribution of trans-membrane voltage on bacteria has also been simulated. Suitable parameters can form perforation with enough size to kill bacteria without overmuch energy consumption and thermogenesis. Experimental studies also obtain similar results as the simulation. Based on the simulation, optimal sterilization device and electric parameters can be achieved.

Low voltage pulse injection test of a single-stage 1 MV prototype induction voltage adder cell.

Low voltage pulse injection tests have been done on a 1 MV prototype induction cell. The tests mainly aim at measuring azimuthal uniformity of feed currents and evaluating cell electrical parameters. Tests are conducted under two cases that the cell is fed by a single pulse and two pulses, respectively. The results indicate that, in the case of the single-point feed, the best current uniformity with an azimuthal variation of 19.3% is acquired when azimuthal lines connect to cathode plates with lower half circumferences. In the case of two-pulse synchronous feed, the current uniformity becomes better, and a current with an azimuthal variation of 11.8% is achieved. Moreover, the effects of asynchronous feed on current uniformity are also experimentally investigated. The results imply that, for given injecting pulses with the duration of 70-80 ns, a time deviation less than 30 ns could be acceptable, without obvious degradation on the feed uniformity and current addition. In addition, the influences of the current uniformity and amounts of feed pulses on cell equivalent inductances are evaluated. The experiment results show that, the equivalent inductance would nearly keep a value of 130 nH as the current variation is less than 50%. However, the extreme asymmetry of feed currents or the increase of amounts of feed pulses would produce additional inductance.

Design of a Two Levels Self-Integrating Capacitive Divider

A ims . A kind of two levels self-integrating capacitive voltage divider is designed to online measure high voltage nanosecond pulse. Methods. The circuit principle of capacitive divider is analyzed, and the theoretical voltage ratio is calculated. The calibration is carried out by using a low voltage pulse source. The divider with a primary voltage divide ratio 601 has been tested to frequency response exceed 1GHz. The second voltage divide ratio can be set flexibly using different ratio attenuators according to the different voltage level of the source. Result. Finally, this divider is used in measuring 600kV high voltage pulse source, and the experimental results show that the present coaxial capacitive divider is reliable and satisfactory for the measuring requirement in time domain and frequency domain.

Transdermal transport pathway creation: Electroporation pulse order

In this study we consider the physics underlying electroporation which is administered to skin in order to radically increase transdermal drug delivery. The method involves the application of intense electric fields to alter the structure of the impermeable outer layer, the stratum corneum. A generally held view in the field of skin electroporation is that the skin’s drop in resistance (to transport) is proportional to the total power of the pulses (which may be inferred by the number of pulses administered). Contrary to this belief, experiments conducted in this study show that the application of high voltage pulses prior to the application of low voltage pulses result in lower transport than when low voltage pulses alone are applied (when less total pulse power is administered). In order to reconcile these unexpected experimental results, a computational model is used to conduct an analysis which shows that the high density distribution of very small aqueous pathways through the stratum corneum associated with high voltage pulses is detrimental to the evolution of larger pathways that are associated with low voltage pulses.

Safe and efficient local gene delivery into skeletal muscle via a combination of Pluronic L64 and modified electrotransfer

Efficient DNA electrotransfer into muscles can be achieved by combining two types of electronic pulses sequentially: short high-voltage (HV) pulse for the cell electropermeabilization and long low-voltage (LV) pulse for the DNA electrophoresis into cells. However, the voltages currently applied can still induce histological and functional damages to tissues. Pluronic L64 has been considered as a molecule possessing cell membrane-disturbing ability. For these reasons, we hope that L64 can be used as a substitute for the HV pulse in cell membrane permeabilization, and a safe LV pulse may still keep the ability to drive plasmid DNA across the permeabilized membrane. In this work, we optimized the electrotransfer parameters to establish a safe and efficient procedure using a clinically applied instrument, and found out that the critical condition for a successful combination of electrotransfer with L64 was that the injection of plasmid/L64 mixture should be applied 1 h before the electrotransfer. In addition, we revealed that the combined procedure could not efficiently transfer plasmid into solid tumor because the uncompressed plasmid may rapidly permeate the leaky tumor vessels and flow away. Altogether, the results demonstrate that the combined procedure has the potential for plasmid-based gene therapy through safe and efficient local gene delivery into skeletal muscles.

Skin Electroporation of a Plasmid Encoding hCAP-18/LL-37 Host Defense Peptide Promotes Wound Healing

Host defense peptides, in particular LL-37, are emerging as potential therapeutics for promoting wound healing and inhibiting bacterial growth. However, effective delivery of the LL-37 peptide remains limiting. We hypothesized that skin-targeted electroporation of a plasmid encoding hCAP-18/LL-37 would promote the healing of wounds. The plasmid was efficiently delivered to full-thickness skin wounds by electroporation and it induced expression of LL-37 in the epithelium. It significantly accelerated reepithelialization of nondiabetic and diabetic wounds and caused a significant VEGFa and interleukin (IL)-6 induction. IL-6 was involved in LL-37-mediated keratinocyte migration in vitro and IL-6 neutralizing antibodies delivered to mice were able to suppress the wound healing activity of the hCAP-18/LL-37 plasmid. In a hindlimb ischemia model, electroporation of the hCAP-18/LL-37 plasmid increased blood perfusion, reduced muscular atrophy, and upregulated the angiogenic chemokines VEGFa and SDF-1a, and their receptors VEGF-R and CXCR-4. These findings demonstrate that a localized gene therapy with LL-37 is a promising approach for the treatment of wounds.

Investigation of Low-Voltage Pulse Parameters on Electroporation and Electrical Lysis Using a Microfluidic Device With Interdigitated Electrodes

Electroporation (EP) of biological cells leads to the exchange of materials through the permeabilized cell membrane, while electrical lysis (EL) irreversibly disrupts the cell membrane. We report a microfluidic device to study these two phenomena with low-voltage excitation for lab-on-a-chip (LOC) applications. For systematic study of EP, we have employed a quantification metric: flow Index (FI) of EP. Simulation and experimental results with the microfluidic device containing interdigitated, coplanar, integrated electrodes to electroporate, and rapidly lyse biological cells are presented. H&E stained human buccal cells were subjected to various pulse magnitudes, pulsewidths, and number of pulses. Simulations show that an electric field of 25kV/cm with a 20V applied potential produced 1.3 (°)C temperature rise for a 5s of excitation. For a 20V pulse-excitation with pulse-widths between 0.5 to 5s, EL was observed, whereas for lower excitations, only EP was observed. FI of EP is found to be a direct function of pulse magnitudes, pulsewidths, and numbers of pulses. To release DNA from nucleus, excitation-pulses of 5s were required. Quantification of EP would be useful for systematic study of EP toward optimization with various excitation pulses, while low-voltage requirement and high yield of EP and EL are critical to develop LOC for drug delivery and cell-sample preparation, respectively.