electronics

Article
Validation of a SPICE Model for High Frequency
Electroporation Systems
Paulius Butkus *, Sonata Tolvaišienė and Sebastjanas Kurčevskis
Department of Electrical Engineering, Faculty of Electronics, Vilnius Gediminas Technical University,
Sauletekio al. 11, Vilnius 10221, Lithuania; sonata.tolvaisiene@vgtu.lt (S.T.); sebastjanas.kurcevskis@vgtu.lt (S.K.)
* Correspondence: paulius.butkus@vgtu.lt; Tel.: +370-680-51-641




Received: 29 May 2019; Accepted: 20 June 2019; Published: 23 June 2019


Abstract: In this paper, we present an analysis and a validation of a simulation program with integrated
circuit emphasis (SPICE) model for a pulse forming circuit of a high frequency electroporation system,
which can deliver square-wave sub-microsecond (100–900 ns) electric field pulses. The developed
SPICE model is suggested for use in evaluation of transient processes that occur due to high frequency
operations in prototype systems. A controlled crowbar circuit was implemented to support a variety
of biological loads and to ensure a constant electric pulse rise and fall time during electroporation to be
independent of the applied buffer bioimpedance. The SPICE model was validated via a comparison
of the simulation and experimental results obtained from the already existing prototype system. The
SPICE model results were in good agreement with the experimental results, and the model complexity
was found to be sufficient for analysis of transient processes. As result, the proposed SPICE model
can be useful for evaluation and compensation of transient processes in sub-microsecond pulsed
power set-ups during the development of new prototypes.

Keywords: electroporation; SPICE model; pulsed electric field; high-voltage generator

1. Introduction
High-pulsed electric fields (PEFs), used to induce an electric field across a cell membrane, have
many applications [1–6]. However, PEF application in electroporation has mostly been researched in
the field of medicine and the food industry [7,8]. The electroporation phenomenon, induced by a PEF,
is driven by the reorientation of lipids and formation of pores in the cell membrane, resulting in an
increase in cell permeability [9]. Thus, higher cell absorption of drugs and genes can be achieved as a
result of increased molecular transportation via the cell membrane [10]. For this reason, the use of
electroporation is gaining momentum, and new medical applications are emerging at an increasing rate.
In order to trigger the intended electroporation processes, the typical transmembrane voltage
threshold is 200 mV or higher [11,12]. This is acquired through accumulation of sufficient charge
during electric pulsing, which allows for the membrane permittivity to change. After the threshold is
reached, the electroporation phenomenon is initiated. As a result, electroporation is a pulse-dependent
phenomenon [13,14], which can be investigated using electric circuit models. A biological cell can be
represented as an electrical resistor–capacitor (RC) circuit, wherein the RC value represents the cell
and the suspension charging constant.
The electroporation process is only initiated effectively after the induced steady state
transmembrane voltage is reached. Hence, the desired effect and efficiency of the electroporation
process for medical applications depends on specific pulse parameters, such as the electric pulse
duration (varies from tens of ns to ms range), pulse number, repetition frequency, direction of the
generated pulse (monopolar, bipolar), electric field amplitude (varies from millivolt up to several
kilovolt range) and shape of the pulse [14–20].

Electronics 2019, 8, 710; doi:10.3390/electronics8060710 www.mdpi.com/journal/electronics

Electronics 2019, 8, 710 2 of 10

Usually, a trade-off between the PEF amplitude and duration is made under a certain pulse
repetition number, but the proper PEF application protocol is influenced by the cell type in general [21,22].
For instance, for mammalian cells, a well-established protocol is to apply a burst of microsecond
pulses (e.g. 100 µs × 8) in the kV/cm PEF strength range [22,23]. However, in recent years, numerous
researchers have started to focus on shorter (sub-microsecond) but higher intensity (range of kV)
PEF pulses [24–26]. The reasoning for this is based on the ability to use generated pulse energy
more efficiently—due to reduced thermal influence—as well as to have the possibility of bypassing
the plasma membrane to affect a cell’s inner organelles directly [27–29]. Additional interest into the
use of high-voltage sub-microsecond PEFs lies in the possibility of inducing cell apoptosis [30,31].
However, one of the most particular focuses is in biomedical applications (e.g., improvement of
electrochemotherapy and tissue ablation protocols) [22].
Different PEF waveforms have been applied in electroporation techniques, such as square-wave
(rectangular), exponential decay, triangular and sinusoidal waveforms. However, it has been
demonstrated that square-wave and exponential decay pulses have higher efficiency in comparison
with other types of pulses [32]. As a result, a majority of PEF generators produce pulses in one of these
two types of waveforms. However, further studies have reported that square-wave pulses are more
efficient during the application of high intensity (tens to hundreds of kV/cm) nanosecond PEFs, due
to its enhanced control of the pulse energy [33]. This is an important aspect for the rapidly growing
microfluidics-based electroporation technique [34], wherein planar electrode structures have proved to
be advantageous [34,35].
Most pulsed electric field generators available in the market for commercial use are made for
specific PEF application, with predefined pulsed parameters and limited control possibility. These
specifications limit the possibilities for application in the electroporation research area, where higher
flexibility for setting different protocols is required. The development of a flexible, high-frequency
electroporation system, aligned with advanced planar electrodes, could contribute toward further
investigation of biomedical techniques with medical applications.
Therefore, in this study, we analyzed a simulation program with integrated circuit emphasis
(SPICE) model of a pulse forming circuit of a high frequency electroporation system, which was capable
of delivering square-wave sub-microsecond (100–900 ns) electric field pulses. A SPICE model, which
was introduced in previous study [36], was further developed and a validation test was performed via
a comparison of simulation and experimental results obtained from the prototype system. The SPICE
model results were in good agreement with the experimental results. In addition, the complexity of
the developed SPICE model was sufficient for analysis of transient processes. Our simulation model
contributes to the prototyping of flexible electroporation systems, and can be applied for evaluation
and compensation of transient processes in the emerging sub-microsecond pulsed power set-ups by
reducing the time and costs associated with the prototyping phase.

2. PEF Generator Specifications

As mentioned, the PEF parameter requirements for an electroporation system depend on the
intended biological processes, which are meant to be induced by high electric fields in biological
objects. The pulse parameters, such as the electric field strength, pulse amplitude, duration, waveform
and repetition frequency, are selected according to the requirements of the experiments. Therefore,
in this study, considering the development in electroporation research specified above, we were aiming
to develop and present an electroporator system capable of generating single and burst PEF pulses
of variable duration in the range of 100 ns to 1 ms. This range of controllable PEF pulse duration
would ensure that the device was flexible and therefore applicable in a wide area of electroporation
research activities.
As mentioned, use of a short pulse duration means a trade-off with PEF amplitude must be made.
A short pulse duration (in range of 100 ns) increases the required electric field strength needed to
reach the transmembrane voltage threshold due to the amount of energy involved. Consequently, the
Electronics 2019, 8, 710 3 of 10

application of sub-microsecond pulses requires an electric field strength of at least 30 kV/cm. In the
case wherein standard 1 mm electroporation cuvettes are used, a high-voltage power supply (in the
range of at least 2–3 kV) has to be applied to reach a sufficient electric field strength. However, as the
overall aim of a variable and controllable PEF typically remains, the pulse forming circuit should be
able to deliver adjustable power PEF pulses with a voltage level ranging from 0 kV to 3 kV.
Additionally, it is expected that a biological load will vary from 50 Ω to 500 Ω during experimental
applications. Meanwhile, the peak value of a pulsed electric current should not exceed 60 A at the
lowest expected load and at the maximum voltage range. This requirement is determined by the
current handling capability and induced Joule heating on biological object, which negatively impact
the electroporation process [37]. Therefore, high-energy pulses are rarely used.
Furthermore, since the efficiency of some treatment applications can be dependent on the pulse
frequency, the device should be capable of altering the pulse frequency over an extensive range. Thus,
we decided that the PEF frequency range had to be adjustable from 1 Hz to 3.5 MHz. Moreover, in order
to have good control of the pulse energy, the square-wave pulse form was preferred, and repeated
pulses had to have identical form. Based on all the requirements specified above, a summary of the
pulse forming circuit requirements is provided in Table 1.

Table 1. Pulse forming circuit requirements.

Parameter Value
Pulse amplitude 1 V to 3 kV
Electric field strength ≤30 kV/cm
Number of pulses 1–99
Repetition frequency 1 Hz to 3.5 MHz
Pulse duration 100 ns to 1 ms
Current 0–60 A

3. SPICE Model of the PEF Generator

3.1. Design of the Pulse Forming Circuit

Designing a flexible pulse forming circuit for an electroporation system is a challenging and
multidisciplinary task. It requires application of numerical methods to investigate the electrical
circuit’s transient processes and parasitic elements, such as the contact capacitance, resistance of the
transmission line and stray inductance. Inappropriate application of compensation circuits may lead
to the appearance of overvoltage, overcurrent or waveform distortion, as well as over-damped or
under-damped responses. These would result in a poorly performing pulse forming circuit. Hence,
to compensate for the transient processes that occur during turn-on and turn-off time, which appear
due to parasitic circuit parameters, proper compensation circuits must be developed [38]. Moreover,
load matching and driving circuit topologies for the semiconductor switches should be solved.
In order to investigate and design the measures needed to compensate the parasitic circuit
parameter’s influence on the PEF generator, the commercially free LTSpice simulation software was
used to create a SPICE model of the pulse forming circuit. The developed SPICE model of the
pulse forming circuit for the PEF generator is presented in Figure 1. Considering the high frequency
requirements described in previous section, the pulse forming circuit switching control was limited to
the high-frequency semiconductor MOSFET switches, which are suitable to ensure high dV/dt.
Synchronized control of the MOSFET switches was ensured via the usage of a microprocessor,
which also provided the user with the ability to precisely define the electric field strength, duration
range, pulse number and frequency. Thus, the PEF generator was able output either a single short
square-wave pulse or a number of multiple pulses. As seen in Figure 1, the pulse forming circuit
consisted of three main groups:
Electronics 2019, 8, 710 4 of 10

• A pulse driving circuit unit, which was composed of an ADUM4223 isolated gate driver (Analog
Devices, Norwood, USA). This gate driver is designed for higher switching speeds and provides
reliable control over the switching characteristics of MOSFET switches.
• A pulse power circuit, which was composed of six high speed and low capacitance C2M0080120D
silicon carbide power MOSFET switches (SW1–SW6, CREE, Durham, USA), manufactured by
CREE. This type of switch ensures high system switching frequency.
Electronics 2019, 8, x FOR PEER REVIEW 4 of 10
• A high-voltage DC power supply supported by the capacitor array, for energy accumulation.

Control part
Microprocessor

DR4 DR3 DR6 DR5 DR2 DR1 Power

supply
RDR3 RDR5 RDR2 RDR1 Cln
RDR3 RDR6
SW4 SW3 SW6 SW5 SW2 SW1

CS4 RS4 CS3 RS3 CS6 RS6 CS5 RS5 CS2 RS2 CS1 RS1 V

D4 D3 D6 D5 D2 D1

RLoad Crowbar
RLIM RLIM
CP LP

Cuvette

Figure 1. The simplified simulation program with integrated circuit emphasis (SPICE) pulse forming
Figure 1. The simplified simulation program with integrated circuit emphasis (SPICE) pulse forming
circuit for the pulsed electric field (PEF) generator.
circuit for the pulsed electric field (PEF) generator.
The SW1–SW6 switches were employed to form the square-wave pulses. For the compensation
Synchronized
circuit, control
snubber diodes D1 ofto the
D6 MOSFET switches was
were implemented ensuredtovia
in parallel theMOSFET
each usage of switch
a microprocessor,
to provide
which also provided the user with the ability to precisely define the electric
reverse voltage protection. In addition, energy absorbing RC snubber circuits were connected field strength, durationin
range, pulse number and frequency. Thus, the PEF generator was able output
parallel to each MOSFET switch in order to eliminate sudden voltage surges in the pulse forming either a single short
square-wave
circuit createdpulse or inductance
by stray a number of multiple
and to ensurepulses.
MOSFET As seen in Figure 1, This
synchronization. the pulse
type offorming circuit
compensation
consisted of three main groups:
circuit ensures proper surge voltage damping during turn-on and turn-off time, thereby protecting the
• A pulse
power MOSFET driving circuitand
switches unit, which
other was composed
devices of an ADUM4223 isolated gate driver (Analog
in its vicinity.
Devices, Norwood, USA). This gate driver is
The semiconductor switches were driven by commerciallydesigned for available
higher switching
two-channelspeeds and provides
MOSFET drivers
reliable control over the switching characteristics of MOSFET switches.
DR1–DR6, ensuring the galvanical isolation of the control circuit. In the SPICE model, to imitate the
• A pulse
galvanic power circuit,
isolation, the MOSFETwhich was composed
switches were of six high speed
connected to theand low
pulse capacitance
control C2M0080120D
unit throughout the
silicon carbide power MOSFET switches (SW1–SW6, CREE, Durham, USA), manufactured by
power transformers (transformation ratio equal to 1) and current limiting resistors, RDR . The pulse
CREE.
driving Thisunit
circuit type(control
of switchpartensures
shownhigh system
in Figure switching frequency.
1) represents the up-to-4 A output, current-isolated,
• A high-voltage DC power supply supported by the capacitor array, for energy accumulation.
half-bridge gate ADUM4223 driver, providing control over the MOSFET switches with a frequency up
to 3.5The
MHz, SW1–SW6 switches
output drive were
voltage employed
ranging fromto4.5form
V tothe square-wave
18 V, pulses.
and an isolation For the compensation
capability up to 5 kV. As
circuit, snubber diodes D1 to D6 were implemented in parallel to each MOSFET switch to provide
a result, the driving circuit ensured precise and reliable control of the switching characteristics.
reverse voltage protection. In addition, energy absorbing RC snubber circuits were connected in
The crowbar circuit, which operated in synchronous mode with SW3 and SW4, was introduced to
parallel to each MOSFET switch in order to eliminate sudden voltage surges in the pulse forming
ensure short pulse fall time and independence from the load (a more detail operational performance
circuit created by stray inductance and to ensure MOSFET synchronization. This type of
is explained in the following section). To ensure a good match of switching characteristics between
compensation circuit ensures proper surge voltage damping during turn-on and turn-off time,
the SPICE model and the PEF generator prototype system, the SPICE model of C2M0080120D silicon
thereby protecting the power MOSFET switches and other devices in its vicinity.
carbide power MOSFET switch from CREE (USA) was used during the simulations, because the same
The semiconductor switches were driven by commercially available two-channel MOSFET
type of switch was used in the prototype system. This switch was also used for SPICE model validation.
drivers DR1–DR6, ensuring the galvanical isolation of the control circuit. In the SPICE model, to
Each C2M0080120D switch was capable of handling voltages up to 1.7 kV and currents up to 72 A.
imitate the galvanic isolation, the MOSFET switches were connected to the pulse control unit
Furthermore, all of the MOSFET switches were paired in series to be able to withstand commutation
throughout the power transformers (transformation ratio equal to 1) and current limiting resistors,
voltages up to 3 kV.
RDR. The pulse driving circuit unit (control part shown in Figure 1) represents the up-to-4 A output,
current-isolated, half-bridge gate ADUM4223 driver, providing control over the MOSFET switches
with a frequency up to 3.5 MHz, output drive voltage ranging from 4.5 V to 18 V, and an isolation
capability up to 5 kV. As a result, the driving circuit ensured precise and reliable control of the
switching characteristics.
The crowbar circuit, which operated in synchronous mode with SW3 and SW4, was introduced
Electronics 2019, 8, 710 5 of 10

The parasitic snubber circuit capacitance (CS ) and the parasitic circuit inductance (Lp ) of the
printed circuit board (PCB) circuit were, respectively, represented by a capacitor and an inductor
connected to the pulse forming circuit in series. In addition, the load resistance (RLOAD ) and parasitic
load capacitance (CP ) imitated the cell and the suspension charging constant. In order to simulate real
electroporation experiment parameters, where low conductivity buffers are used, the load of the SPICE
model was varied from 50 Ω to 500 Ω. The main parameters of the created SPICE model of the PEF
generator are shown in Table 2.

Table 2. Pulse forming circuit requirements.

Parameter Min. Value Max. Value Final Value

Snubber resistance (RS ) 0.1 Ω 20 Ω 0.5 Ω
Snubber capacitance (CS ) 200 pF 900 pF 500 pF
Load (RLOAD ) 50 Ω 500 Ω 50–500 Ω
Parasitic load capacitance (CP ) 10 pF 100 pF 50 pF
Parasitic circuit inductance (Lp ) 1 µH 5 µH 1.2 µH

In order to suppress the transient processes of the pulse forming circuit, the parameters shown in
Table 2 were swept from the minimum to the maximum values. The impact of each parameter was
evaluated to not only find the optimal RC snubber circuit parameters, but also to have a good match
with the measurement values obtained from the actual PEF generator prototype system.

3.2. The Pulse Forming Circuit Operational Description

Switches SW1–SW2 and SW3–SW4 belonged to the main power line (see Figure 1) and were
responsible for commutation of the power source voltage over the load. When these MOSFET switches
were turned on, a high-voltage pulse was over the load. Consequently, all of these switches operated
in a synchronized mode and, thus, were opened and closed at the same time. The pulse rise time was
dependent on the switching characteristics of the switches, as well as the parasitic circuit inductance
and capacitance.
The switches SW5–SW6 represented a controllable crowbar circuit, which was triggered by
DR5–DR6 at the same time as when the pulse power circuit switches in the pairs SW1–SW2 and
SW3–SW4 were closed. In this way, the crowbar circuit could be opened to create a new path for the
high current to flow to the ground, avoiding a path through the cuvette—since this path was closed at
the same time by SW1–SW2 and SW3–SW4. As a result, the pulse fall time became independent of the
load, but was influenced by the switching characteristics of the switches and the parasitic parameters
of the circuit.
The resulting pulse on the load of 100 Ω with the implemented crowbar circuit is shown in
Figure 2. The SPICE model simulation results indicated that the crowbar circuit reduced the pulse
fall time from 30 ns to 18 ns; the controllable and synchronized crowbar circuit minimized the fall
time of the pulse. It removed the characteristic load-dependent tail and provided an opportunity for
sub-microsecond pulse generation with a high impedance buffer.
In this way, the presented pulse forming circuit offered flexibility for different experimental
applications, while the SPICE model itself was suitable for the electrical pulse forming circuit transient
processes and analysis of the parasitic elements.
As will be demonstrated below, the SPICE model was also able to optimize prototype development
and reduce the time and cost needs during the prototype development phase.
Electronics 2019, 8, x FOR PEER REVIEW 6 of 10
Experiment VS modeling

4.0
Simulations on different loads with crowbar
Electronics 2019, 8, 710 3.5 Simulations on different loads without crowbar 6 of 10
Electronics 2019, 8, x FOR PEER REVIEW Measurement on the load cuvette with crowbar 6 of 10
Experiment VS modeling
3.0
4.0 RLOAD = 500 
2.5

Voltage (kV) Voltage (kV)

Simulations on different loads with crowbar
RLOAD = 300 
3.5 Simulations on different loads without crowbar
2.0  the load cuvette with crowbar
RLOAD = 150 on
Measurement
3.0
1.5 R = 150 
RLOAD = 500 LOAD
2.5 RLOAD = 300 
1.0 RLOAD = 300 
2.0 RLOAD = 150 
0.5
1.5
0.0 RLOAD = 150 

1.0 RLOAD = 300 

-0.5
150 200 250 300 350 400 450 500
0.5
Time (ns)
0.0
Figure 2. The comparison of pulse fall time with and without the crowbar circuit under different
-0.5
loads. 150 200 250 300 350 400 450 500
Time (ns)
As will be demonstrated below, the SPICE model was also able to optimize prototype
Figure
development2. The
Figure 2. Thecomparison
and comparison
reduce
ofofpulse
the timepulsefall time
andfall
cost
with
time andand
with
needs
without the crowbar
duringwithout
circuitcircuit
the crowbar
the prototype
underunder
development
different loads.
different
phase.
loads.
3.3. The Impact of the RC Snubber Circuit
3.3. The Impact of the RC Snubber Circuit
As will line
Parasitic be inductance
demonstrated and below,
parasiticthe
loadSPICE modelhave
capacitance was also able
a negative to optimize
impact prototype
on the performance
of Parasitic
development
pulse forming line
and inductance
reduce
circuits andthecantimeand parasitic
and cost
impose load capacitance
needsprocesses
transient during the have a negative
prototype development
of overvoltage impact
and overcurrent.phase. on the
In addition,
performance of pulse forming circuits and can impose transient processes of
other undesirable pulse distortions, like circuit ringing, can be triggered. As a result, the generation overvoltage and
overcurrent.
3.3.3 The
of kV Impact
squareIn of
addition, other undesirable
the RC pulses
electric Snubber Circuit
for pulse distortions,
PEF electroporation like circuit
treatment ringing,iscan
applications be triggered.
complicated andAsa
amultidisciplinary
result, the generation of 3 kV square electric pulses for PEF electroporation treatment applications
Parasitic lineengineering
inductancetask, and which cannot
parasitic loadbe capacitance
solved without have a proper set-upimpact
a negative of RC snubber
on the
is complicated
circuits. Figure and
3 a multidisciplinary
presents the parasitic engineering
circuit task, which
inductance cannot
influence on be solved
the pulse, without
which a proper
resulted in
performance of pulse forming circuits and can impose transient processes of overvoltage and
set-up of
overvoltageRC snubber circuits. Figure 3 presents the parasitic circuit inductance influence on the pulse,
overcurrent.during turn-on
In addition, and
other
R=150 ohm
turn-off of the
undesirable semiconductor
pulse MOSFET
distortions, like circuit switches.
ringing, can be triggered. As
which resulted in overvoltage during turn-on and turn-off of the semiconductor MOSFET switches.
a result, the generation of 3 kV square electric pulses for PEF electroporation treatment applications
4.0
is complicated and a multidisciplinary engineering task, which cannot Lp = 1.00 be
H solved without a proper
3.5
set-up of RC snubber circuits. Figure 3 presents the parasitic circuit inductance L p
= 1.25 H influence on the pulse,
R=150 ohm Lp = 1.75 H
which resulted in overvoltage 3.0 during turn-on and turn-off of the semiconductor MOSFET switches.
Lp = 3.25 H
4.0
2.5 Lp = 5.00 H
Lp = 1.00 H
Voltage (kV) Voltage (kV)

3.5
2.0 Lp = 1.25 H
Lp = 1.75 H
3.0
1.5 Lp = 3.25 H
2.5 Lp = 5.00 H
1.0
2.0
0.5
1.5
0.0
1.0
-0.5
0 50 100 150 200 250 300
0.5
Time (ns)
0.0
Figure 3.
Figure The output
3. The output voltage
voltage pulse
pulse on
on the
the load
load of
of 150 Ω without
150 Ω without the
the snubber
snubber and
and crowbar
crowbar circuits.
circuits.
-0.5
0 50 100 150 200 250 300
One of
One ofthe
themain
maintasks during
tasks the development
during the development of the sub-microsecond square-wave
of the sub-microsecond PEF generator
square-wave PEF
Time (ns)
was to compensate the transient processes in the pulse forming circuit during
generator was to compensate the transient processes in the pulse forming circuit during the rise and the rise and fall time
of the
fall timegenerated
Figure 3. The
of the pulse.
output To
generated solvepulse
voltage
pulse. Tothis issue,
on
solvethe
this RC
load snubber
of
issue, 150 circuits
RCΩ snubber
without thewere implemented
snubber
circuits were crowbarinto
andimplemented the
circuits.
intopulse
the
forming circuit and connected in parallel with the semiconductor MOSFET
pulse forming circuit and connected in parallel with the semiconductor MOSFET switches. In switches. In addition,
the RC One of the main tasks
snubber during the development of the of the sub-microsecond
switches.square-wave PEF
addition, the RCcircuits
snubber provided
circuits synchronization
provided synchronization series-connected
of the series-connected In the prototype
switches. In the
generator wasphase,
development to compensate
thephase,
designtheoftransient processes in the pulsecan
forming circuit during the rise and
prototype development the optimal
design of RCoptimal
snubber RCcircuits
snubber be a time
circuits canconsuming and
be a time consuming costly
fall time
task if theofdifferent
the generated
set-ups pulse.
of RC To solve this
snubber issue,
circuits are RC snubber circuits
experimentally were
tested implemented
directly on the into the
prototype
and costly task if the different set-ups of RC snubber circuits are experimentally tested directly on the
pulse forming
system. circuithand,
On the other and this
connected
task caninbeparallel
more easilywithsolved
the semiconductor
via application MOSFET
of a properswitches.
simulation In
addition, the RC snubber circuits provided synchronization of the series-connected switches. In the
prototype development phase, the design of optimal RC snubber circuits can be a time consuming
and costly task if the different set-ups of RC snubber circuits are experimentally tested directly on the
Electronics 2019,
Electronics 8, 710
2019, 8, x FOR PEER REVIEW 7 7ofof
10 10

prototype system. On the other hand, this task can be more easily solved via application of a proper
model (such as a SPICE model). However, the developed SPICE model has to be validated to be trusted
simulation model (such as a SPICE model). However, the developed SPICE model has to be validated
for use in the design phase of any new prototype system.
to be trusted for use in the design phase of any new prototype system.
4. SPICE Model Validation
4. SPICE Model Validation
The prototype of a flexible PEF generator addressing all of the requirements discussed above has
alreadyThe
beenprototype
developed of for
a flexible
the studyPEF of generator addressing
electroporation all of research
in previous the requirements
[26,39]. Indiscussed
this study,above
the
has already been developed for the study of electroporation in previous research
same PEF generator was used to verify the SPICE model presented above by performing a comparison [26,39]. In this study,
ofthe
the same PEF and
numerical generator was used
experimental to verify
results of the the SPICE
output model presented above by performing a
pulse.
comparison of the numerical and experimental results
As a first step of validation, a single compensated output pulse of the output
on thepulse.
load of 100 Ω was simulated
with theAsSPICE
a first step under
model of validation,
different RCa single
snubber compensated outputand
circuit parameters pulse on the with
compared loadtheof measured
100 Ω was
simulated with the SPICE model under different RC snubber circuit parameters
single output pulse of the PEF generator prototype system. The PEF generator output signal on the and compared with
the
100 Ωmeasured
load cuvette single
wasoutput
measured pulse of the
using thePEF
1:100generator
probe and prototype
a DPO4034 system.
digitalThe PEF generator
oscilloscope output
(Tektronix,
Beaverton, OR, USA). To capture the output waveform, the experimental results were recordeddigital
signal on the 100 Ω load cuvette was measured using the 1:100 probe and a DPO4034 as a
oscilloscope
mean (Tektronix, Beaverton, OR, USA). To capture the output waveform, the experimental
of five measurements.
results were recorded
The results as a mean are
of the comparison of five measurements.
presented in Figure 4, which shows the measured output pulse
of the developed PEF generator prototype systeminand
The results of the comparison are presented Figure
two4,simulation
which shows the measured
curves with swept output pulse
snubber
of the developed PEF generator prototype system and two simulation curves
resistance, RS . The single 2.5 kV output pulse waveform at the minimum duration of the existing PEF with swept snubber
resistance,
generator wasRused
S. The single 2.5 kV output pulse waveform at the minimum duration of the existing PEF
for this comparison. As seen in Figure 4, the pulse duration (measured at 50% of the
pulse amplitude)used
generator was was 90fornsthis comparison.
(±10%). It was As seenby
limited in the
Figure 4, the pulse
MOSFET switchesduration (measured
switching at 50% of
characteristics
thethe
and pulse amplitude)
parasitic parameters wasof90thens (±10%).
circuit. It time
A rise was oflimited by athe
28 ns and fallMOSFET
time of 18switches switching
ns was measured
characteristics and the parasitic parameters of the circuit. A rise time of
(10–90% of the pulse amplitude). The measured values matched well with the simulation results, 28 ns and a fall time of 18 ns
was measured (10–90% of the pulse amplitude).
underlining the influence of the crowbar circuit on output voltage. The measured values matched well with the
simulation results, underlining the influence of the crowbar circuit on output voltage.
4.0
Measurement on the load cuvette
3.5 Simulations with Rs=10 , Cs=500 pF
Simulations with Rs=0.5 , Cs=500 pF
3.0
28 ns 18 ns
2.5
Voltage (kV)

90% 90%
2.0

1.5 90 ns
50%
1.0

0.5
10% 10%

0.0

-0.5
25 50 75 100 125 150 175 200 225 250
Time (ns)

Figure The
4. 4.
Figure Thecomparison ofof
comparison simulation and
simulation experimental
and results
experimental ofof
results a single electrical
a single pulse
electrical waveform
pulse waveform
generated by the PEF generator.
generated by the PEF generator.

In addition, as seen in Figure 4, the snubber resistance of 10 Ω and the snubber capacitance of
In addition, as seen in Figure 4, the snubber resistance of 10 Ω and the snubber capacitance of
500 pF resulted in a faster turn-off time and better minimization of the reverse overvoltage; however,
500 pF resulted in a faster turn-off time and better minimization of the reverse overvoltage; however,
the snubber resistance of 0.5 Ω with 500 pF snubber capacitance fit better with the measured signal.
the snubber resistance of 0.5 Ω with 500 pF snubber capacitance fit better with the measured signal.
These results suggest that the RC snubber circuits used in the prototype system were not optimally
These results suggest that the RC snubber circuits used in the prototype system were not optimally
designed. In any case, the implemented RC circuit helped to suppress a transient process and limit the
designed. In any case, the implemented RC circuit helped to suppress a transient process and limit
reverse overvoltage during the fall time of the pulse, while the new SPICE model was proved to be
the reverse overvoltage during the fall time of the pulse, while the new SPICE model was proved to
sufficient for the prototyping of new PEF generators.
be sufficient for the prototyping of new PEF generators.
 Electronics
Electronics 2019,
2019, 8, x FOR PEER REVIEW
8, 710 8 8ofof
1010

As an additional step for SPICE model validation, a comparison of the simulated and generated
As of
series anPEF
additional
pulses step for SPICE model
was performed at the validation, a comparison
highest frequency ofand
(3.5 Hz) the simulated
voltage (3 and generated
kV) output. The
series
PEF of PEF pulses
generator wassignal
output performed
on theat100
theΩhighest frequency
load cuvette was (3.5
alsoHz) and voltage
measured using(3 kV)
the output.
1:100 probeThe
and
PEF
thegenerator
DPO4034output
digital signal
oscilloscope 100 Ω load Beaverton,
on the(Tektronix, cuvette wasOR, also measured
USA). usingthe
To capture theoutput
1:100 probe and
waveform,
the
theDPO4034 digital results
experimental oscilloscope
were(Tektronix,
recorded Beaverton,
as a meanOR, of USA). To capture the output
five measurements. waveform,
The SPICE model
the experimental
simulations results
were were recorded
performed under theas asame
meanload
of five measurements.
and supply voltage Theconditions
SPICE model simulations
as the measured
were performeddata
experimental under the same
in order to beload
able and supply the
to compare voltage conditions
numerical aswith
results the measured experimental
the measured values. The
data in order
results to validation
of this be able to compare
step are the numerical
presented results5.with the measured values. The results of this
in Figure
validation step are presented in Figure 5.

Figure 5. Simulation and experimental results of the PEF generator’s high-voltage sub-microsecond
Figure
output 5. Simulation
pulses andofexperimental
on the load results offrequency
100 Ω with repetition the PEF generator’s high-voltage sub-microsecond
up to 3.5 MHz.
output pulses on the load of 100 Ω with repetition frequency up to 3.5 MHz.
In Figure 5 we can see the measured 3 kV output pulse of the developed PEF generator. It appears
that pulses with a5duration
In Figure we can of
see80the
ns measured
(measured 3onkV 50% of thepulse
output pulse of
amplitude) and a rise
the developed PEFand fall time It
generator.
ofappears
25 ns and 18pulses
that ns, respectively, were generated.
with a duration The developed
of 80 ns (measured on 50% prototype system
of the pulse of the PEF
amplitude) andgenerator
a rise and
was
fallcapable
time of of
25generating pulses
ns and 18 ns, with a frequency
respectively, up to 3.5The
were generated. MHz. In Figure
developed 5, it can system
prototype be seenofthat
thethe
PEF
simulation
generatorresults of the SPICE
was capable model were
of generating in good
pulses withagreement
a frequencywith
upthe measured
to 3.5 MHz. In output signal
Figure 5, itof thebe
can
prototype
seen thatsystem. Thus, ourresults
the simulation modelof may
thebe considered
SPICE modelsufficient
were in for
goodanalyzing
agreementtransient processes
with the measuredin
electroporation
output signal systems and can be
of the prototype used in
system. the prototype
Thus, our modeldesign
may bephase of new sufficient
considered PEF generators.
for analyzing
transient processes in electroporation systems and can be used in the prototype design phase of new
5.PEF
Conclusions
generators.
In this paper, we presented an analysis and a validation of a SPICE model for a pulse forming
5. Conclusions
circuit of a high frequency electroporation system, which was controlled by high-frequency MOSFET
switchesIn and
this was
paper,capable of delivering
we presented square-wave
an analysis and a sub-microsecond
validation of a SPICE PEF pulses. TheaSPICE
model for model
pulse forming
was developed
circuit to frequency
of a high be used forelectroporation
the evaluation system,
of pulsewhich
formingwascircuit transient
controlled processes, which
by high-frequency occur
MOSFET
asswitches
a consequence of high frequency operation, and for the optimization of RC snubber
and was capable of delivering square-wave sub-microsecond PEF pulses. The SPICE model circuit design in
future PEF generators.
was developed A controlled
to be used crowbar circuit
for the evaluation wasforming
of pulse implemented
circuittotransient
support processes,
a variety ofwhich
biological
occur
loads and to ensure a constant rise and fall time during electroporation, independent
as a consequence of high frequency operation, and for the optimization of RC snubber circuit design of the applied
buffer bioimpedance.
in future The SPICE
PEF generators. model wascrowbar
A controlled validatedcircuit
via a comparison of simulation
was implemented and experimental
to support a variety of
results obtained
biological loadsfrom
andthe prototype
to ensure system.rise
a constant Theand
SPICE
fall model resultselectroporation,
time during were in good agreement
independentwithofthe
the
experimental results and the model complexity was proved to be sufficient for
applied buffer bioimpedance. The SPICE model was validated via a comparison of simulation and analysis of transient
processes. The results
experimental results of this study
obtained fromandthe
theprototype
validatedsystem.
SPICE model will contribute
The SPICE to thewere
model results evaluation
in good
and compensation
agreement with theof experimental
the transient processes
results and inthe
sub-microsecond
model complexity pulsedwaspower
provedset-ups, as well asfor
to be sufficient
analysis of transient processes. The results of this study and the validated SPICE model will
Electronics 2019, 8, 710 9 of 10

the development of new prototypes. Future work should continue with development and validation
of the planar electrodes simulation model, which would allow for simulation of a wider range of
experimental conditions in electroporation research.

Author Contributions: Conceptualization, P.B. and S.T.; methodology, P.B.; software, P.B.; validation, P.B., S.T.
and S.K.; formal analysis, P.B.; investigation, P.B.; data curation, P.B.; writing—original draft preparation, P.B.;
writing—review and editing, P.B., S.T. and S.K.; visualization, P.B.; supervision, S.T.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Akiyama, H.; Sakugawa, T.; Namihira, T.; Takaki, K.; Minamitani, Y.; Shimomura, N. Industrial applications
of pulsed power technology. IEEE Trans. Dielectr. Electr. Insul. 2007, 14, 1051–1064. [CrossRef]
2. Jang, S.-R.; Ryoo, H.-J.; Ok, S.-B. Pulsed-power system for leachate treatment applications. J. Power Electron.
2011, 11, 612–619. [CrossRef]
3. Barba, F.J.; Parniakov, O.; Pereira, S.A.; Wiktor, A.; Grimi, N.; Boussetta, N.; Saraiva, J.A.; Raso, J.;
Martin-Belloso, O.; Witrowa-Rajchert, D.; et al. Current applications and new opportunities for the use of
pulsed electric fields in food science and industry. Food Res. Int. 2015, 77, 773–798. [CrossRef]
4. Schoenbach, K.H.; Katsuki, S.; Stark, R.H.; Buescher, E.S.; Beebe, S.J. Bioelectrics-new applications for pulsed
power technology. IEEE Trans. Plasma Sci. 2002, 30, 293–300. [CrossRef]
5. Gómez, B.; Munekata, P.E.S.; Gavahian, M.; Barba, F.J.; Martí-Quijal, F.J.; Bolumar, T.; Campagnol, P.C.B.;
Tomasevic, I.; Lorenzo, J.M. Application of pulsed electric fields in meat and fish processing industries: An
overview. Food Res. Int. 2019, 123, 95–105. [CrossRef]
6. Sitzmann, W.; Vorobiev, E.; Lebovka, N. Applications of electricity and specifically pulsed electric fields in
food processing: historical backgrounds. Innov. Food Sci. Emerg. Technol. 2016, 37, 302–311. [CrossRef]
7. Yarmush, M.L.; Golberg, A.; Serša, G.; Kotnik, T.; Miklavčič, D. Electroporation-Based technologies for
medicine: Principles, applications, and challenges. Annu. Rev. Biomed. Eng. 2014, 16, 295–320. [CrossRef]
[PubMed]
8. Hart, F.X.; Palisano, J.R. The application of electric fields in biology and medicine. In Electric Field; InTech
Open: London, UK, 2018.
9. Tsong, T.Y. Electroporation of cell membranes. Biophys. J. 1991, 60, 297–306. [CrossRef]
10. Haberl, S.; Miklavcic, D.; Sersa, G.; Frey, W.; Rubinsky, B. Cell membrane electroporation-part 2: The
applications. IEEE Electr. Insul. Mag. 2013, 29, 29–37. [CrossRef]
11. Teissié, J.; Rols, M.P. An experimental evaluation of the critical potential difference inducing cell membrane
electropermeabilization. Biophys. J. 1993, 65, 409–413. [CrossRef]
12. Murovec, T.; Sweeney, D.C.; Latouche, E.; Davalos, R.V.; Brosseau, C. Modeling of Transmembrane potential
in realistic multicellular structures before electroporation. Biophys. J. 2016, 111, 2286–2295. [CrossRef]
[PubMed]
13. Kotnik, T.; Pucihar, G.; Rebersek, M.; Miklavcic, D.; Mir, L.M. Role of pulse shape in cell membrane
electropermeabilization. Biochim. Biophys. Acta 2003, 1614, 193–200. [CrossRef]
14. Pucihar, G.; Krmelj, J.; Reberšek, M.; Napotnik, T.B.; Miklavčič, D.; Reberšek, M.; Napotnik, T.B.; Miklavčič, D.
Equivalent pulse parameters for electroporation. IEEE Trans. Biomed. Eng. 2011, 58, 3279–3288. [CrossRef]
[PubMed]
15. Rebersek, M.; Faurie, C.; Kanduser, M.; Corović, S.; Teissié, J.; Rols, M.-P.; Miklavcic, D. Electroporator with
automatic change of electric field direction improves gene electrotransfer in-vitro. Biomed. Eng. Online 2007,
6, 25. [CrossRef] [PubMed]
16. Kotnik, T.; Kramar, P.; Pucihar, G.; Miklavcic, D.; Tarek, M. Cell membrane electroporation—part 1: The
phenomenon. IEEE Electr. Insul. Mag. 2012, 28, 14–23. [CrossRef]
17. Krassowska, W.; Filev, P.D. Modeling electroporation in a single cell. Biophys. J. 2007, 92, 404–417. [CrossRef]
18. Kotnik, T.; Pucihar, G.; Miklavčič, D. Induced Transmembrane voltage and its correlation with
electroporation-mediated molecular transport. J. Membr. Biol. 2010, 236, 3–13. [CrossRef]
Electronics 2019, 8, 710 10 of 10

19. Towhidi, L.; Kotnik, T.; Pucihar, G.; Firoozabadi, S.M.P.; Mozdarani, H.; Miklavčič, D. Variability of the
minimal transmembrane voltage resulting in detectable membrane electroporation. Electromagn. Biol. Med.
2008, 27, 372–385. [CrossRef]
20. Schoenbach, K.H.; Pakhomov, A.G.; Semenov, I.; Xiao, S.; Pakhomova, O.N.; Ibey, B.L. Ion Transport into
cells exposed to monopolar and bipolar nanosecond pulses. Bioelectrochemistry 2015, 103, 44–51. [CrossRef]
21. Dean, D.A. Cell-specific targeting strategies for electroporation-mediated gene delivery in cells and animals.
J. Membr. Biol. 2013, 246, 737–744. [CrossRef]
22. Novickij, V.; Ruzgys, P.; Grainys, A.; Šatkauskas, S. High frequency electroporation efficiency is under
control of membrane capacitive charging and voltage potential relaxation. Bioelectrochemistry 2018, 119,
92–97. [CrossRef] [PubMed]
23. Ruzgys, P.; Novickij, V.; Novickij, J.; Šatkauskas, S. Nanosecond range electric pulse application as a non-viral
gene delivery method: Proof of concept. Sci. Rep. 2018, 8, 15502. [CrossRef] [PubMed]
24. Garner, A.L.; Caiafa, A.; Jiang, Y.; Klopman, S.; Morton, C.; Torres, A.S.; Loveless, A.M.; Neculaes, V.B.
Design, characterization and experimental validation of a compact, flexible pulsed power architecture for ex
vivo platelet activation. PLoS ONE 2017, 12, e0181214. [CrossRef] [PubMed]
25. Tan, Y.; Yang, H.; Wu, J.; Yang, X.; Zhang, Y.; Zeng, G.; Zhang, X. The sub-microsecond pulser applied for
electroporation effect. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi 2012, 29, 615–619. [PubMed]
26. Novickij, V.; Zinkevičienė, A.; Perminaitė, E.; Čėsna, R.; Lastauskienė, E.; Paškevičius, A.; Švedienė, J.;
Markovskaja, S.; Novickij, J.; Girkontaitė, I. Non-invasive nanosecond electroporation for biocontrol of
surface infections: An in vivo study. Sci. Rep. 2018, 8, 14516. [CrossRef] [PubMed]
27. Deng, J.; Schoenbach, K.H.; Buescher, E.S.; Hair, P.S.; Fox, P.M.; Beebe, S.J. The effects of intense submicrosecond
electrical pulses on cells. Biophys. J. 2003, 84, 2709–2714. [CrossRef]
28. Schoenbach, K.; Hargrave, B.; Joshi, R.; Kolb, J.; Nuccitelli, R.; Osgood, C.; Pakhomov, A.; Stacey, M.;
Swanson, R.; White, J.; et al. Bioelectric effects of intense nanosecond pulses. IEEE Trans. Dielectr. Electr.
Insul. 2007, 14, 1088–1109. [CrossRef]
29. Beebe, S.J.; Chen, Y.-J.; Sain, N.M.; Schoenbach, K.H.; Xiao, S. Transient features in nanosecond pulsed electric
fields differentially modulate mitochondria and viability. PLoS ONE 2012, 7, e51349. [CrossRef] [PubMed]
30. Pakhomova, O.N.; Gregory, B.W.; Semenov, I.; Pakhomov, A.G. Two modes of cell death caused by exposure
to nanosecond pulsed electric field. PLoS ONE 2013, 8, e70278. [CrossRef]
31. Beebe, J.S. Mechanisms of nanosecond pulsed electric field (NsPEF)-induced cell death in cells and tumors. J.
Nanomed. Res. 2015, 2, 00016. [CrossRef]
32. Miklavcic, D.; Towhidi, L. Numerical study of the electroporation pulse shape effect on molecular uptake of
biological cells. Radiol. Oncol. 2010, 44, 34–41. [CrossRef] [PubMed]
33. Kandušer, M.; Miklavčič, D. Electroporation in Biological Cell and Tissue: An Overview; Springer: Berlin,
Germany, 2009; pp. 1–37.
34. Geng, T.; Lu, C. Microfluidic electroporation for cellular analysis and delivery. Lab Chip 2013, 13, 3803–3821.
[CrossRef] [PubMed]
35. Novickij, V.; Tabašnikov, A.; Smith, S.; Grainys, A.; Novickij, J.; Tolvaišienė, S.; Markovskaja, S. Feasibility of
parylene coating for planar electroporation copper electrodes. Mater. Sci. 2017, 23, 93–97. [CrossRef]
36. Butkus, P.; Kurcevskis, S.; Tolvaisiene, S.; Grainys, A. Computer model of the high frequency up to 30
KV/Cm electric field generator. In Proceedings of the 2017 5th IEEE Workshop on Advances in Information,
Electronic and Electrical Engineering (AIEEE), Riga, Latvia, 24–25 November 2017; pp. 1–3.
37. Pliquett, U.; Nuccitelli, R. Measurement and simulation of joule heating during treatment of B-16 Melanoma
tumors in mice with nanosecond pulsed electric fields. Bioelectrochemistry 2014, 100, 62–68. [CrossRef]
[PubMed]
38. Novickij, V.N.; Stankevic, V.; Grainys, A.; Novickij, J.; Tolvaisiene, S. Microsecond electroporator optimization
for parasitic load handling and damping. Elektron. Elektrotechnol. 2015, 21, 40–43.
39. Novickij, V.; Grainys, A.; Butkus, P.; Tolvaišienė, S.; Švedienė, J.; Paškevičius, A.; Novickij, J. High-frequency
submicrosecond electroporator. Biotechnol. Biotechnol. Equip. 2016, 30, 607–613. [CrossRef]

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