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A review of electrochemical impedance as a tool

Cite this: DOI: 10.1039/d3an01423a
for examining cell biology and subcellular
mechanisms: merits, limits, and future prospects
Seyedyousef Arman,a,b Richard D. Tilley a,c
and J. Justin Gooding *a,b
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Herein the development of cellular impedance biosensors, electrochemical impedance spectroscopy,

and the general principles and terms associated with the cell–electrode interface is reviewed. This family
Received 18th August 2023, of techniques provides quantitative and sensitive information into cell responses to stimuli in real-time
Accepted 22nd November 2023
with high temporal resolution. The applications of cell-based impedance biosensors as a readout in cell
DOI: 10.1039/d3an01423a biology is illustrated with a diverse range of examples. The current state of the field, its limitations, the
rsc.li/analyst possible available solutions, and the potential benefits of developing biosensors are discussed.

1. Introduction continuous monitoring of cells, with high temporal resolution,

as they respond to stimuli.
The biggest challenge to deciphering the language of biologi- Electrochemical impedance spectroscopy (EIS) has been
cal cells arises from the complex nature of the cells and count- widely utilized in different areas of chemical science.25–28 Over
less interactions in different cell lines.1 The development of the past few decades, there has been a growing interest among
screening methods capable of unveiling the myriad of ques- researchers in using EIS for investigations in cell biology.29–36
tions arising from the interaction of biological cells with drugs The interest in cell biology was stimulated by the 1984 report
has been an area of intensive research in biology, pharmacy, by Giaever and Keese on what they called electric cell–substrate
biochemistry, biophysics, and engineering.1–4 Most cell-based impedance sensing (ECIS). The technique enables continuous,
assays currently rely on identifying and visualization of cellular quantitative and qualitative evaluation of live cells in biological
regulating molecules using fluorescent dyes or genetically media.37 The central idea is that the cell membranes can be
encoded fluorescent indicators.5–8 considered as insulating entities such that culturing and/or
Even though these fluorescent techniques have had incubating cells on an electrode surface can function as a
immense success, label-free technologies are emerging as a barrier that impedes the flow of electrical signals.38 By moni-
promising alternative in providing continuous, real-time toring the time-dependent alterations in impedance, encom-
insights into the physiological function of cells.9–15 The passing resistance and capacitance, the method is able to
central concept that renders label-free biosensors attractive is provide information on cellular processes occurring directly on
the potential for a low cost and the continuous record of cells the electrode surface. Researchers may determine and charac-
in response to their environment.16 As these non-invasive terize the differences in cell states by comparing the impe-
assays omit the labelling step and use native cells, question dance profiles recorded at various times or under different
over the addition of detection reagents is not required. experimental circumstances. Impedance signals may serve as
There are a different label-free approaches to monitor cells indicators for several biological phenomena, including cell
such as resonant waveguide grating (RWG), surface plasmon attachment, spreading,38 the extent of cell adherence to the
resonance (SPR), acoustic biosensors, electrochemilumine- modified electrode surface39 micromotion,40 proliferation,
scence and electrochemical impedance-based biosensors.17–24 migration,41 tumour metastasis,42 cell signalling,43 and barrier
Of these, electrochemical impedance methods are ideal for the function of the cells.44 As cell barrier behaviour is linked to
intracellular interactions inside the cell, impedance can be uti-
lised for the purpose of analysing both intracellular and inter-
a
School of Chemistry, The University of New South Wales, Sydney, New South Wales cellular level. Accordingly, impedance cell biosensing has been
2052, Australia. E-mail: [email protected] successfully established to investigate chronic diseases includ-
b
Australia Centre for Nanomedicine, The University of New South Wales, Sydney,
ing epithelial and endothelial barrier function,45,46 cancer,47
New South Wales 2052, Australia
c
Electron Microscope Unit, Mark Wainwright Analytical Centre, The University of cytotoxicity,48 infectious diseases49 and the response of cells to
New South Wales, Sydney, New South Wales 2052, Australia drugs.50 This review sets out to explain the main concepts

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behind impedance, the pros and cons of cell-based impedance

biosensor performance and future research opportunities in
the application of impedance biosensors in a variety of biologi-
cal contexts.

2. Principles and terms in

electrochemical impedance
Fig. 2 The alternating current exhibits a sinusoidal response to the
spectroscopy applied voltage waveform at an angular frequency of ω in a linear
system.
In electrical circuits, resistance and impedance are commonly
understood as elements that impede the flow of electrical
current. They could be evaluated as the ratio between the levels. Consequently, applying a low-amplitude voltage at a fre-
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input voltage and the resulting current that passes through an quency of ω leads to the generation of an alternating current
electrical circuit. Despite the similarity in definition and com- with a similar frequency and a phase shift. Both current and
putation between impedance and resistance values, their fun- ! !
voltage can be represented with separate vectors (VðtÞ or IðtÞ)
damental origins are different. The concept of resistance is that rotate at the same frequency. According to the data pre-
based on the utilisation of a DC signal, whereas impedance is sented in Fig. 2, it can be observed that the current and poten-
dependent upon applying an AC signal.51 The impedance tial exhibit a phase difference, which results in the possibility
could be derived by applying either voltage or current to the of vector separation by a constant phase angle (φ) as the
working electrode, and according to the transfer function: vectors undergo rotation.55 Therefore, the expression for the
Ð1 current vector of length I0 is as follows:
V ðsÞ vðtÞest dt
ZL ¼ HðSÞ ¼ ¼ Ð01 st dt
ð1Þ
IðSÞ 0 iðtÞe !
IðtÞ ¼ I0 sinðωt þ φÞ: ð4Þ
where ZL is the Laplace impedance function, s is the Laplace
frequency, V(s) and I(s) are the Laplace transforms of voltage The principle of resistance, which adheres to Ohm’s law
and current–time function.52,53 Impedance spectroscopy, in and remains unaffected by frequency, is applicable to AC cir-
contrast to conventional electrochemical techniques, rep- cuits when the vectors of current and voltage are in phase with
resents data in the frequency domain rather than time, poten- each other (as depicted in Fig. 3). The current and voltage in
tial, or current.54 Given the excitation of the electrochemical vector notation for a pure resistance (R) subjected to a sinusoi-
system through a potential sine-wave, it is possible to represent dal potential, in accordance with Ohm’s law, can be expressed
the voltage as a rotating vector (generally referred as a phasor), as follows, with a phase angle of zero:55,56
as depicted in Fig. 1, in which its length is the amplitude (V0),
and the frequency of rotation is ω as follows: !
! VðtÞ V0
IðtÞ ¼ ¼ sinðωtÞ ð5Þ
! R R
V ðtÞ ¼ V0 sinðωtÞ: ð2Þ
The angular frequency (ω) could be written as a function of ! !
conventional frequency ( f ): V ðtÞ ¼ IðtÞR: ð6Þ

ω ¼ 2πf : ð3Þ In addition, when a sinusoidal current is applied to a pure

capacitance, the expressions for charge and current can be rep-
It is noteworthy that the excitation signal of an AC source
typically exhibits a low magnitude, owing to the linear nature
of the current–overpotential relationship at low overpotential

Fig. 1 A sinusoidal voltage is being applied and rotating at an angular Fig. 3 The current generated when a sinusoidal potential is applied to a
frequency of ω. pure resistance.

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dV Table 1 Different ways of impedance presentation

resented as Q = CV or I ¼ C . The phasor notation represen-
dt
tation of the current is as follows: Result Related
description parameters Definition Illustration
! V0  π
IðtÞ ¼ CdðV0 sinðωtÞÞ=dt ¼ ωCV0 cosðωtÞ ¼ sin ωt þ Nyquist plot Z(ω) = Zre − iZim Impedance ZImaginary vs. ZReal
Xc 2 Bodé module |Z| = (Zre)2 + (Zim)2 Magnitude |Z| vs. frequency
ð7Þ of impedance
Bodé phase tan φ = Zim/Zre = Phase angle Φ vs. frequency
Xc is the capacitance reactance. plot Xc/R = 1/ωRc
Fig. 4 shows that the phasor notation for the current and
voltage has a phase angle shift of 2π , and the vectors are situ-
ated within the same plane. Given that the phase angles for a quency. Impedance spectroscopy typically involves frequency
pure resistance and pure capacitance are 0 and π/2, respect- sweeps ranging from several kHz to 10−1 or 10−3 Hz. It is feas-
ively, it is reasonable to anticipate an intermediate phase for a ible to restrict the frequency range to a limited number of fre-
combination of resistance and capacitance.55 Therefore, the quencies or a singular frequency mode with the aim of acquir-
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phase shift (φ) in relation to the voltage waveform as well as ing specific information during a certain time. Under such cir-
the amplitude have an impact on the current–time function. cumstances, it is possible to measure the temporal variation of
The use of complex arguments has been suggested to facilitate resistance or capacitance. The utilisation of alternating current
mathematical procedures. In the field of complex analysis, it is signals exhibiting varying frequencies, as opposed to direct
common practise to designate the terms along the Y-axis that current waveforms, may yield more significant insights at each
pertain to capacitance behaviour as the imaginary part. This is frequency interval, encompassing the double-layer capacitance
denoted by the imaginary unit, which is represented by the of the electrochemical system being examined.57
pffiffiffiffiffiffiffi
symbol j ¼ 1. Conversely, the terms along the X-axis, which
relate to resistance behaviour, are typically regarded as the real
part.54 Accordingly, the potential in phasor notation could be 3. The beginning of impedance
expressed as follow: analysis in cell biosensing
! !
V ðtÞ ¼ jXc IðtÞ: ð8Þ Giaever and Keese37 conducted a study aimed at comprehend-
When examining the application of a sinusoidal potential ing the impacts of electromagnetic fields on cells, which led to
to a series circuit consisting of resistance and capacitance, it the development of cellular impedance biosensors.58 For the
can be observed that the overall potential is equivalent to the first time, they applied a DC potential to mammalian cells by
combined potential drop across both the resistor and the placing them between two electrodes. However, the ions ema-
capacitor. The voltage can be determined by analysing the nating from the surface of the electrodes resulted in the death
current through the impedance vector, which is represented by of the cells. The technique of impedance sensing for cellular
the equation (Z = R − iXc) as follow:55 analysis was developed by utilising a single-frequency AC
signal instead of a DC signal with a relatively high frequency
! ! ! ! !
V ¼ VR þ VC ¼ I ðR  iXc Þ ¼ I Z: ð9Þ in an effort to impede the generation of ion products on the
surface. Despite this, identical impedance responses were pro-
Various plots can be used to demonstrate the impedance duced on a large working electrode regardless of whether cells
outcomes. Table 1 summarises the pertinent parameters, defi- were present or not. This observation suggested that the
nitions, and modes of illustration.57 Table 1 presents a culture solution resistance was far greater compared to that of
description of the magnitude of the impedance and the corres- the layer of cells present on the electrode, and it dominated
ponding phase angle, both of which are dependent on the fre- the overall results of the test. Since the resistance of the
medium is in line with the solid–liquid interface impedance,
the impedance response of the cells on the surface depends
extremely on the dimension of the sensing (working)
electrode.40,58 In general, the total impedance in biological
sensing employing impedance is governed by the sensing elec-
trode impedance, hence counter electrode impedance must be
lower compared to the sensing electrode.51,52,57
In the very first arrangement proposed by Giaever and
Keese, the ratio of the area of the working electrode (WE) to
the counter electrode (CE) was approximately 1 : 1500.37 The
initial setup for cell sensing in a Petri dish is illustrated in
Fig. 5. This configuration comprises a large gold electrode
Fig. 4 The current generated when a sinusoidal potential is applied to (2 cm2) functioning as a counter and four tiny golden electro-
a pure capacitance. des (3 × 10−4 cm2) serving as sensing electrodes. They used a

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potential to mitigate the impact of solution resistance within

the system. Reducing the sensing area is a commonly
employed strategy to enhance the sensitivity of detecting
alterations in microelectrodes covered by cells.66,70 However, it
has been reported66 that the ultra-sensitivity of impedance bio-
sensors may lead to the instability of electrical signals, which
can result in issues with data replication.
Stolwijk et al.,66 demonstrated the significance of electrode
configuration by employing three different electrode configur-
ations (8W1E, 8W10E, and 8W10E+) to examine the barrier
function of HDMEC cells, as depicted in Fig. 6. According to
Fig. 5 (a) The initial electrode setup for use as a biosensor in cellular Fig. 6a and b, it can be observed that the sensing area of 8W1E
analysis is shown. It illustrates the schematic of the gold pattern on the (5 × 10−5 cm2) is 10 times smaller than that of 8W10E. As a
bottom of the Petri dish with a large gold rectangle as the counter elec- result, the number of cells that can be monitored using 8W1E
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trode and four smaller microelectrodes as sensing electrodes. Copper

is limited, ranging from 20 to 100 cells depending on the cell-
wires are soldered to all electrodes. (b) This step involves enclosing the
four microelectrodes using a glass coverslip while maintaining a precise line. Despite the fact that the resistance associated with the
distance of 100 µm between the edge of the coverslip and the four 8W1E setup exhibits a greater value than that of 8W10E (as
microelectrodes. (c) The image illustrates the application of molten red depicted in Fig. 6d), the sensitivity (as illustrated in Fig. 6e)
wax as an insulating material for the gold–copper contacts. (d) The pro- remains nearly identical. Increasing the number of cells under
vided image represents the initial electrode arrangement used for con-
observation may be advantageous in reveal certain biological
ducting electric cell–substrate impedance sensing. Images (a–c) are
reproduced from ref. 37 PNAS. The figure (d) is reprinted from ref. 58 responses exhibited by the cells. Regarding this matter, it can
with permission from Springer; permission obtained through Copyright be observed that 8W10E+ (as shown in Fig. 6c) has the capacity
Clearance Center, Inc. to accommodate a greater number of cells due to the presence
of 40 circular sensing electrodes, each arranged on an interdi-
gitated gold pattern. Based on the observations made in
Fig. 6d and e, it is evident that despite the different configur-
lock-in amplifier to provide an AC signal of 100 mV between ation of 8W10E as compared to 8W10E+, both exhibit compar-
the small working electrodes and large counter electrode, and able electrical characteristics. The observed behaviour can be
the constant current between the electrodes was reported to be
about 1 µA.37,58,59
The literature reveals that various electrode configurations
have been developed to ensure that the impedance of the
sensing electrode dominates that of the solution
resistance.60–66 Due to the requirement of a significantly
smaller sensing electrode in comparison to the counter elec-
trode, the monitoring capacity of cells is restricted to a limited
number at any given time.58,66 Parallel utilisation of multiple
sensing electrodes has been a common practise in most
configurations.58,60–66 An approach that could be employed
involves the use of multiplexers that facilitate switching
between different electrodes. As a result, the number of cells
under analysis increases by a factor equal to the number of
electrodes used.60,67
Important progress has also been made in the field of elec- Fig. 6 The impact of electrode configurations on the sensitivity of elec-
trical impedance cell biosensing, with the development of trical cellular impedance biosensor analysis. (a–c) Schematic drawings
interdigitated electrodes in 1996 68 as a novel electrode of microelectrode patterns on the bottom of a single well belonging to
arrangement for monitoring cell activity at the surface. commercial 8W1E plate, which includes 8 wells and 1 × 250 µm circular
sensing electrode in diameter, versus a large counter electrode per well,
Applied Biophysics (under the trade name ECIS), ACEA (under
8W10E plate, including 8 wells and 10 × 250 µm circular sensing elec-
the trade name xCELLigence), and MDS Sciex (under the trade trodes in diameter, versus a large counter electrode per well, 8W10E+
names Roche and Cell Key) have all commercialised various plate, including 8 wells and 20 × 250 µm circular sensing electrodes in
electrode configurations and formats (8, 16, 96, and 384 diameter, versus 20 × 250 µm circular counter electrodes in diameter on
wells).69 All commercial devices utilised the identical approach an interdigitated pattern per well. (d) Resistance response of cells at
different frequencies for 8W1E, 8W10E, and 8W10E+. (e) Analysis of the
of using small sensing electrodes combined with a large
sensitivity of cell sensing for the three electrode configurations by
counter electrode or interdigitated electrode pattern. The sen- measuring Rcell-covered/Rcell-free. The figure reprinted from ref. 66 with
sitivity of a systems can vary depending on the electrode con- permission from Springer; permission obtained through Copyright
figuration used.70 The configuration of the electrode has the Clearance Center, Inc.

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attributed to similar contributions from both the sensing and rc and current to be a constant value in the Z direction (verti-
counter electrodes. cal) that flows between cell–substrate interface (Fig. 7b), as
Gold has been a desirable electrode material for many years well as some simple assumptions about potential:40,79
due to its many useful properties, including being biocompati-
ble, noble, and highly conductive. However, the growing inter- d2 V 1 dV
þ  ϒ 2V þ β ¼ 0 ð10Þ
est in cell imaging has been a significant motivator for transi- dr 2 r dr
tioning towards the utilisation of transparent and conductive
material electrodes.71–75 In recent years, there has been an  
ρ 1 1
increasing interest among researchers in the field regarding ϒ ¼
2
þ ð11Þ
h Zn Zm
the utilisation of ITO. However, ITO exhibits lower conductivity
compared to gold and will fail to provide the same level of sen-
 
sitivity as gold, though it is feasible to tailor ITO to meet par- ρ Vn Vm
B¼ þ ð12Þ
ticular requirements for enhancing electrical cell sensing h Zn Zm
sensitivity.
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The enhancement of the electrical properties of ITO can be where the variables Vn and Vm represent the bare electrode
achieved through the use of surface treatment. According to lit- potential and the extracellular potential in the solution.
erature,76 the use of interdigitated ITO electrodes coated with Meanwhile, ρ and h denote the specific resistivity of the
IrOx can result in a reduction of interfacial impedance in com- medium in which the cells are cultured and the average cell–
parison to the use of pure ITO. This is perhaps the only study substrate distance, respectively. Additionally, Zn and Zm corres-
so far that shows how to achieve a higher sensitivity with ITO pond to the specific impedance of the solid–liquid interface
to detect electrical behaviour of cells; in this case breast cancer without cells and the specific impedance between two cellular
cells (MCF-7). membranes, respectively. The Bessel equation is a linear
second-order ordinary differential equation expressed in the
form x2y″ + xy′ + (x2 − V2)y = 0. The parameter V is commonly
4. Modelling and calculation referred to as the order of the Bessel equation. Therefore, the
Bessel function offers a linear solution to this differential
The principle of electrochemical impedance cell–substrate equation, and the following equation can be used to calculate
sensing relies on applying a relatively low AC potential the specific impedance of the electrode covered by a cell:40,80
between a sensing and counter electrode.77,78 This low voltage
2 3
is non-invasive and has been extensively shown in scientific lit- Zm
erature to be far below the level at which it might impact cellu- 1 1 6 Zn Zn þ Zm  7
¼ 64 þ  7 ð13Þ
lar processes or induce electrostimulation. It should be noted Zc Zn Zn þ Zm iϒrc I0 ðϒ rc Þ 1 1 5
þ 2Rb þ
that in impedance analysis, any voltage that can generate a 2 I1 ðϒ rc Þ Zn Zm
constant current of 1 µA is typically considered acceptable and
safe for cell research. Fig. 7a demonstrates that the current where I0 and I1 are modified Bessel functions of the first kind
pffiffiffiffiffiffiffi
path is extremely sensitive to the applied frequency.79 Giaever (order 0 and 1) with complex arguments and i ¼ 1, the
and Keese proposed the following differential equation by modified Bessel functions are expressed to expand the
assuming cells to be circular cross section with a diameter of complex variable as follows:

sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
 ffi rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ρ 1 1 1 1
ϒ rc ¼ rc þ ¼α þ ð14Þ
h Zn Zm Zn Zm

Z m ¼ i=ð2πf ÞðCm =2Þ ð15Þ

where Zn is the specific impedance of the bare electrode

without cells, Zm is the specific impedance between two cellu-
lar membranes in line through a layer of cells. The only para-
meters that might be used for fitting the model are α, Rb, and
Cm since Zn and Zc can be measured empirically and Zm can
Fig. 7 The basic principle of cell–electrode impedance to noninvasively be calculated using a frequency dependent formula. Rb reflects
analyse cellular morphology by applying and maintaining a low current the degree of cell–cell contact, while α indicates the measure
between electrodes and representation of cells on microelectrode. (a)
of space between the cells and the substrate.66 Bessel functions
Different current paths at low frequencies (solid lines) and high frequen-
cies (broken lines). (b) A schematic view of a single cell on working elec-
cannot be solved directly because of the previously mentioned
trode emphasising the average cell–substrate distance and the current connection with the parameter (α, Rb, and Cm), but they may
path. be extracted by curve fitting.81

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5. Application within the framework pathways in G protein-coupled receptors (GPCRs). Fig. 8b, c,
and d depict the impedance profiles pertaining to the Gi, Gq,
of the commercially available devices and Gs signalling pathways, respectively.95,96 Based on the evi-
5.1 Chemical stimulation of receptors dence, we may conclude that activating Gi-coupled receptors
Complex signalling networks are employed by cells to determine raises cellular impedance, whereas activating Gq receptors
and respond to their surrounding environmental changes.82 The results in a similar rise in impedance after a brief drop, and
processing of biological signals involves the identification of activating Gs receptors reduces cellular impedance. The
chemical stimuli through receptors on the cellular membrane.82 capacity to identify and discriminate diverse signalling path-
Upon activation, the receptor facilitates signal transduction to ways within a single assay represents a significant advance-
various cellular components by regulating specific proteins. It is ment. In the past few years, the application of cellular impe-
important to consider that a single receptor has the potential to dance biosensor has become prevalent in tackling issues
trigger numerous signalling pathways.83 Furthermore, the cell related to receptor pharmacology and the development of new
line and agonist play a role in how receptors regulate a signalling therapeutic ligands.97–103
pathway.84 This highlights the challenge of fully comprehending The combined use of electrical impedance and pharmaco-
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the mechanisms underlying receptor activation in living organ- logical inhibitors may shed light on cellular processes, from
isms. Assays that rely on labelling as a means of detecting recep- receptors to signalling molecules. By way of illustration,
tor activation are typically limited by their ability, as they solely Meshki et al.104 used the electrical cellular biosensor to show
monitor a single signalling pathway at any given time.85 Hence, that pre-treatment of HEK293 cells with the ROCK inhibitor
the assessment of an unfamiliar ligand’s binding to a receptor (Y27632) eliminated the blebbing and morphological changes
through conventional assays may necessitate a considerable associated with neurokinin 1 receptor (NK1R) activation,
degree of experimentation.86 leading to their interpretation shown in Fig. 9. These results
G protein activation has been widely recognised as a mecha- were in contrast to earlier studies indicating that the activation
nism that can trigger diverse signalling pathways, leading to of NK1R may result in the activation of the Gq pathway via
alterations in cellular polarity and cytoskeletal reorganisation.87,88 mechanisms including increased intracellular calcium and
The latter produces alterations in the morphology of cell that PKC stimulation. This unexpected result established for the
could be identified through the use of a cellular impedance bio- first time that the NK1R is activated through the Rho/ROCK/
sensor, suggesting the stimulation of either endogenous or MLCK pathway. In another study, Davis et al.105 highlighted
exogenous G protein-coupled receptors (GPCRs).89–92 Electrical the simplicity and speed of impedance cellular assay to study a
impedance assessments allow for continuous monitoring of mul- novel ROCK inhibitory effect.
tiple receptor activities on a single platform, such as those of tyro- Each kind of cell in an organism has its own unique set of
sine kinase receptors (TKRs), nuclear receptors and G protein- signalling pathways to interact with other cells; these pathways
coupled receptors (GPCRs).89,90,93,94
The illustration depicted in Fig. 8a demonstrates the
mechanism of signal transduction via Gq, Gi, and Gs signalling

Fig. 9 A schematic representation of signalling cascade corresponded

to the activation of the NK1R receptor. This model shows that stimu-
Fig. 8 (a) Schematic representation of the main GPCR signalling lation of NK1R receptor results in membrane blebbing. U73122 as a
pathway activated by the four heterotrimeric G proteins. (b–d) Different potent inhibitor of phospholipase C (PLC) blocked the Gq pathway and
specific profiles measured by cellular impedance biosensor corre- releasing intracellular calcium. Nevertheless, membrane blebbing
sponded to Gi, Gq and Gs pathway. The displayed content in figure (b–d) occurred indicating the activation of MLCK/ROCK/Rho through G12/13
reproduced with permission from ref. 84. Copyright 2010 Elsevier; per- signalling pathway. The displayed content reproduced from ref. 104.
mission obtained through Copyright Clearance Center, Inc. Copyright 2009 Elsevier.

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are regulated by membrane receptors. A hallmark of GPCRs,

and a key obstacle for the development of drugs, is the event
of a single receptor activation triggering multiple signalling
pathways. Stallaert et al.86 documented the existence of several
signalling pathways in the impedance profile that are linked to
the triggering of the 2-adrenergic receptor through isoprotere-
nol (ISO). The deconvolution of impedance alterations enabled
the identification of both Gi and Gs cascades, activation of
ERK 12 and cAMP, and an elevation in intracellular calcium
concentration.
According to the literature,84 the existence of multiple
events generating opposing electrical signals may lead to a
lack of alteration in impedance and, consequently, a potential
misinterpretation of the data. An example is the activation of
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the CRF1 receptor, where both Gi and Gs signalling contribute

equally but yield no detectable electrical signal as a result.95 Fig. 10 The equilibrative nucleoside transporter 1 (ENT1) balances the
A specific function of cell membrane receptors is the intracellular and extracellular concentration of adenosine. The presence
of transporters outside the cell stimulates the adenosine receptor (AR)
absorption of photons and the subsequent initiation of photo-
and downstream signalling cascade. Blocking ENT1 using inhibitor led to
sensory signalling within the cell. The activation of photo- the increased extracellular concentration of adenosine and conse-
receptors has been associated with mechanical stresses gener- quently, the rise of adenosine receptor activation. The displayed content
ated on the lipid bilayer of the cellular membrane.106 The reproduced from ref. 109. Copyright 2019 Springer Nature.
possible benefits of using a cellular impedance biosensor
include the detection of photosensory information. Fischer
et al.,107 conducted a study wherein Neuro-2A and HEK293
cells were transfected with TMT-Opsins, and the resulting 5.2 Cell adhesion, immune cell interactions and endothelial
impedance were monitored upon illumination. The results barrier function
indicate that over time, cellular index values rose, up to a dur- Comprehending the processes of cellular attachment and
ation of 4 minutes. Additionally, following the cessation of illu- detachment could shed light on fundamental principles of the
mination, the impedance value returned to the baseline level human biological system, biomedical science, drug therapy,
after a brief period. The investigation also examined the differ- tissue engineering, and, most importantly, the early detection
ences in the kinetic responses of different types of of diseases.110–114 For example, the decreased adhesiveness of
photoreceptors. malignant cells to the extracellular scaffolding has been associ-
In addition to the potential advantages associated with ated with the metastasis and invasiveness of cancerous
impedance for direct binding detection of membrane recep- tumours.115 Cellular impedance biosensors utilise the
tors to a wide variety of chemical and physical environmental dynamic processes of cell adhesion, spreading, and micromo-
factors, cellular impedance measurements may help in the tion to evaluate the electrical signal. These processes cause
understanding of transmembrane transporters. Transporters changes in the surrounding ionic environment on impedance
have a crucial function in both human health and disease,108 microelectrodes. Perhaps this explains why initial studies of
as they are in charge of keeping the pH level stable, maintain- cell impedance focused on how cells adhered to and spread
ing osmotic balance, and participating in signal processing. In across the microelectrode surfaces.
a recent investigation conducted by Vlachodimou et al.,109 Bio-interfaces have a significant impact on the intricate and
whether or not solute carriers (SLCs) might activate G protein- complex process of adhesion. Wegener et al.78 utilised a cell-
coupled receptors (GPCRs) was investigated. The study based impedance biosensor to examine the effects of various
revealed that the inhibition of adenosine transporters resulted extracellular matrix (ECM) proteins on MDCK cell attachment
in an elevation of extracellular adenosine concentration (as and spreading. The parameters derived from the analysis of
illustrated in Fig. 10), which subsequently caused alterations impedance in relation to surface coverage indicate that the
in adenosine receptor signalling. The activation of ligand gate rate of cell spreading is significantly influenced by surface
channels, specifically transient receptor potential ankyrin 1 modification. The differential rates of cellular spreading can
(TRPA1), has been associated with rapid intracellular calcium potentially be explained by distinct mechanisms of interaction
elevation that can persist for a duration of up to 25 minutes. between the cells and bio-interfaces. The study demonstrated
The impedance profile of HEK293 cells expressing TRPA1 in that cell adhesion on a surface modified with laminin was a
response to a specific agonist exhibited a transient reduction result of the interaction with glycolipids, whereas adhesion on
in impedance values, followed by a subsequent return to the a surface modified with fibronectin was attributed to the inter-
initial baseline level.94 An association has been established action with integrin receptors.
between alterations in impedance and a rise in intracellular The implementation of cell-based impedance biosensors
calcium levels. shows promise to differentiate between the specific and non-

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specific adhesion of cells to bio-interfaces, as depicted in isation and intracellular calcium release in HeLa cells upon
Fig. 11. The study conducted by Atienza et al.116 showed that drug administration.
NIH3T3 cells exhibited non-specific adhesion to a surface The concept that the extracellular matrix (ECM) is com-
modified with poly-L-lysine, while they exhibited specific prised of an intricate web of multiple proteins has been widely
adhesion to a surface modified with fibronectin. The study uti- acknowledged.118 Hence, it can be inferred that different
lised an antibody targeting integrin to determine that binding sites are involved in cellular adhesion to the extra-
adhesion events were mediated by integrin receptors present cellular matrix. The study performed by Luong and col-
on the cell membrane. Additionally, the research revealed that leagues119 investigated the role of α2β1 integrin, a cellular
non-specific adhesion was facilitated by charge–charge receptor responsible for the binding of collagen and laminin.
interactions. In this study, human rhabdomyosarcoma cells were employed
Cellular and extracellular matrix (ECM) interactions are to express two variants of the α2β1 integrin receptor: the native
intricate. The diverse spatial configurations of bio-interfaces receptor (RDX2C2) and a mutant receptor lacking the α2
may impact cellular adhesion and associated intracellular domain (RDX2CO). Subsequently, an assessment was con-
signal transduction. Chockalingam et al.117 demonstrated the ducted utilising an impedance biosensor to determine the
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influence of interfacial architecture on the morphology of level of adhesion on micro-electrodes coated with collagen,
cells. A monolayer coating of organo phosphonate was utilised laminin, and fibronectin. The study demonstrated that cells
as a base layer, followed by the sequential construction of bio- that expressed the α2β1 integrin receptor (RDX2C2) exhibited a
interfaces through a stepwise fabrication process. significant increase in adhesion to surfaces that were modified
Subsequently, the ratio of molecules that were attached to the with collagen and laminin. In contrast, the cells that expressed
self-assembled monolayers (SAMs) was regulated prior to the a mutant α2β1 integrin receptor (RDX2CO) demonstrated only
introduction of GRGDC, a cell-adhesive ligand, as the third a slight rise in their adherence to these particular surfaces.
step in the process of cell–molecule coupling. The study Furthermore, it was observed that cells that expressed the α2β1
revealed that the time taken by cells to spread on a precisely integrin receptor (RDX2C2) exhibited a slight elevation in their
defined surface is exquisitely sensitive to the adhesive ligand surface adhesion ability to the modified surface with fibronec-
density on the surface. The impact of surface chemistry on tin. Conversely, cells that expressed the mutant α2β1 integrin
signal processing within cells was revealed in a more recent receptor (RDX2CO) did not demonstrate any adhesion to the
study conducted by the same group.75 The utilisation of a cel- fibronectin-modified surface.
lular impedance biosensor and fluorescence microscopy in a The utilisation of cellular impedance biosensors has the
single device demonstrated that the modification of RGD potential to elucidate a more comprehensive understanding of
spacing on the surface has an impact on cytoskeletal reorgan- cellular behaviour beyond the mere examination of cellular–
ECM interactions. Sawhney et al.,120 investigated the motility
and locomotion of HCT116 cells on surfaces modified with
bovine serum albumin (BSA) and collagen type IV. A notable
characteristic of the resistance profile over time for cells on a
surface modified with collagen type IV was the significant fluc-
tuation of resistance, which suggests the occurrence of micro-
movements of cells on the electrode. In contrast, electrodes
that had undergone surface modification with BSA did not
exhibit any fluctuations, suggesting the absence of micro-
movement. The absence of cell attachment was suggested by
the apparent lack of motility on the BSA modified electrode, as
it is commonly believed that cell motility and attachment to
the surface are biologically linked.
The human immune system is a complex network that
defends the body from disease by deploying specialised
immune cells or proteins to the site of infection. As an
example, the process of leukocyte circulation on endothelial
cells has the potential to trigger the activation of leukocyte
integrins, subsequently leading to their adherence to the endo-
thelial cells. This facilitates the stimulation of actin cytoskele-
Fig. 11 (a) Real-time impedance assessment of NIH3T3 cell adhesion ton reorganisation in endothelial cells, which promotes the
and spreading was conducted using microelectrodes modified with transmigration of leukocytes into the tissue.121 Cellular impe-
poly-L-lysine and fibronectin. (b) Fluorescence images of NIH3T3 cells
dance biosensors are extremely sensitive at detecting altera-
on glass slides modified with poly-L-lysine and fibronectin. The cells
were subjected to staining using rhodamine-phalloidin. The displayed
tions in intercellular junctions and cell–matrix adhesions,
content reproduced from ref. 116. Copyright 2005 Elsevier; permission making them a suitable tool for monitoring the function and
obtained through Copyright Clearance Center, Inc. dysfunction of endothelial barriers. The cellular endothelial

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barrier function is a crucial component in facilitating adaptive function of lung cells. The paper suggests activation of the LPA
immune responses through the process of mobilising immune receptor resulted in an increase in transepithelial resistance,
cells to an injured area. thereby indicating an improvement in barrier function.
According to Wegener et al.122 research, the cAMP-depen- Applying impedance analysis provided evidence that the
dent cellular signalling pathway regulates the defensive func- mechanism of action was through E-cadherin clustering at
tions of choroid plexus epithelial cells, as demonstrated by cell–cell interface. In contrast, the signalling pathway regard-
impedance data. The data analysis revealed that choroid ing TLRs induced epithelial barrier disruption. The transloca-
plexus epithelial cells showed an increased impedance when tion of E-cadherin from the plasma membrane to the cyto-
cAMP was boosted, which suggests a decline in the per- plasm, which was caused by the activation of TLRs, was found
meability of the epithelial barrier function. Similarly, Qiao to be reversed by the activation of LPA, as depicted in Fig. 12.
et al.,123 conducted a study to explore the protective mecha- The intricate arrangement of endothelial cells, which
nism against endothelial barrier dysfunction. The study inves- includes the blood–brain barrier (BBB), are in close proximity
tigated the protective function of human microvascular endo- to one another and form tight junctions between cells in all
thelial cells (HMEC) and pre-treated HMEC (with cAMP-elevat- blood vessels in the brain. The BBB operates as a selective bar-
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ing drugs) to thrombin using a cellular impedance biosensor. ricade that limits the transit of potentially harmful substances
The impedance findings indicate that the reduction in resis- from the bloodstream to the central nervous system, thereby
tance linked to the barrier dysfunction was partially alleviated safeguarding the brain. To date, the most reliable approach for
following the administration of cAMP-elevating medications to analysing the permeability of the blood–brain barrier (BBB)
HMEC. Given that RhoA is involved in the modulation of involves the quantification of transepithelial electrical resis-
HMEC barrier breakdown, it was assumed that the activation tance (TER), which quantifies the restriction of the current
of RhoA could be suppressed by the signalling cascade invol- flow through a monolayer of cells.125 A remarkable character-
ving cAMP-dependent protein kinase (PKA). Therefore, it is istic of the blood–brain barrier is its exceptional capability to
hypothesised that the PKA signalling pathway serves as a resist electrical signals, which is attributed to the presence of
mechanism to mitigate dysfunction of the vascular endothelial tight junctions within the cellular monolayer.126 The impact of
barrier in reaction to diverse inflammatory mediators. cocaine on HIV-1 invasion through the blood–brain barrier
A further assessment of electrical resistance in human was explored by Fiala et al.,127 through the utilisation of cellu-
bronchial epithelial cells (HBEpCs) has revealed the molecular lar electrical impedance. It was shown that the impact of
mechanisms and crosstalk between two different signalling cocaine on brain microvascular endothelial cells (BMECs) was
pathways, as depicted in Fig. 12. He et al.,124 investigated the to cause a disruption of intracellular tight junctions and as a
contribution of the crosstalk between lysophosphatidic acid consequence invasion by HIV-1 was elevated.
receptor (LPA-Rs) and toll-like receptor (TLRs) to the defensive Kataoka et al.,128 conducted a study to examine the inter-
action between arterial endothelial cells and monocytes using
a cellular impedance biosensor and atomic force microscopy.
It was found that the electrical resistance of HUVEC (endo-
thelial cells found in human umbilical veins) exhibited an
instant drop upon being exposed to THP-1, a human monocy-
tic cell line. This behaviour linked to the reduced adhesiveness
and increased deformability of endothelial cells. In a similar
study, Ge et al.129 demonstrated the influence of lipopolysac-
charide (LPS) on the adhesion of leukocytes (U937) to the cells
derived from HUVECs. They showed that the pre-treatment of
HUVECs with LPS caused a decline in adhesion between endo-
thelial cells and substrate that was dependent upon the LPS
dosage. Cellular communication may occur through the
secretion of substances that bind to the receptor of a specific
cell upon activation. Treeratanapiboon et al.130 conducted a
study wherein they triggered human peripheral blood mono-
nuclear cells (PBMC) using membrane-associated malarin
Fig. 12 A schematic illustration of the mechanism that protects the epi-
antigen. This research investigated the effects of activated
thelial barrier disruption induced by lipopolysaccharide (LPS) ligand. The blood cells on the barrier function of porcine brain capillary
LPS activates toll-like receptor 4 (TLRs) regulating mislocalisation of endothelial cells (PBCEC) using a cellular impedance bio-
E-cadherin from cell membrane to cytoplasm. In contrast, the post- sensor. The release of tumour necrosis factor-alpha from
treatment of lysophosphatidic acid (LPA) receptor activates focal
infected blood cells caused increased permeability of the
adhesion kinase (FAK), downstream of protein kinase C (PKC) δ and γ,
that accumulate E (epithelial)-cadherin from the cytoplasm to cell–cell
blood–brain barrier due to damaged tight junctions.
junction and recover the barrier disruption induced by LPS. The dis- Mast cells are a type of primary immune cell that exhibit
played content reproduced from ref. 124. Copyright 2009 Elsevier. rapid responsiveness to allergic stimuli by triggering their

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high-affinity immunoglobulin E (IgE) receptor, commonly D143V_CXCR2 increased at the same rate, suggesting cell pro-
referred to as FcεRI.131 The immune system generates liferation. The WT-CXCR2 exhibited a plateau phase sub-
immunoglobulin E (IgE) as the predominant antibody to safe- sequent to 3000 minutes (50 hours), whereas the
guard the body against allergic reactions. The study conducted D143V_CXCR2 continued to grow throughout the entire dur-
by Abassi et al.132 involved a comparison of the IgE-mediated ation of the experiment, spanning 5000 minutes, without any
signal in RBL-2H3 mast cells using a cellular impedance bio- indication of deceleration. Due to the collapse of contact inhi-
sensor and β-hexosaminidase assay. They identified a corre- bition between the cells, there was unregulated cellular replica-
lation between the electrical signal obtained from impedance tion on top of one another, which led to the development of
and the morphological dynamics, as well as the mediator tumours and a constantly rising impedance profile.
released obtained from the β-hexosaminidase assay. The continuous monitoring of cellular adhesion and move-
ment is critical in understanding cellular processes such as
5.3 Cancer research investigation cellular mitosis, tumour spreading, and cell locomotion in cell
biology.139 Cellular motility is a characteristic associated with
Biosensors that use impedance measurements can differen-
the metastatic potential of malignant cells. Using the impe-
tiate between various human and animal cancer cells by ana-
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dance technique, the motility of cells that are attached to a

lysing their bioelectrical characteristics, such as impedance
surface can be identified and tracked.40,140 Numerous endea-
and capacitance.133–136 This provides an immediate and
vours have been undertaken to establish a correlation between
simple means to gather information about cancer cell line
the adhesion of tumour cells and the formation of metas-
characteristics and the efficacy of anti-cancer drugs.137
tases.141 This phenomenon can be exemplified in human epi-
Impedance data for the normal NIH3T3 mouse fibroblast cell
thelial cells, where the cytoskeletal protein EPLIN (epithelial
line and two transformed cell lines are shown in Fig. 13.138
protein lost in neoplasm) is found. EPLIN is an epithelial
The transformed cell lines were expressed with either a wild-
protein that is not present in tumours and is believed to have
type chemokine receptor (WT-CXCR2) or a receptor with a
a pivotal function in the metastasis of cancer to other
mutation that makes it always active (D143V_CXCR2). The
organs.142 While several theories have been proposed to eluci-
GPCR family includes CXCR2, which typically stimulates cellu-
date the potential involvement of this protein in cellular
lar migration. When this receptor is mutated at position 143,
migration through alterations in cell morphology and
internal signals are constantly triggered, causing normal cells
adhesion, its precise function is not fully understood. Jiang
to transform into malignant ones. Fig. 13 demonstrates that
et al.143 utilised impedance as a tool to investigate how triple-
the resistances corresponding to WT-CXCR2 and
negative breast cancer cells were impacted by EPLIN. The
results indicated a reduction in cell growth, implying that the
molecule may act as a tumour suppressor.
Providing information during therapeutic treatment
enables impedance biosensor to provide us with valuable data
regarding the cell apoptosis, and cancerous cell resistance in
response to chemotherapy. Fig. 14 shows the growth behaviour
of HT-29 colon cancer cells on impedance gold-microelectrode
in response to aspirin. Treating the cell with different concen-
tration of aspirin led to inhibition of cell proliferation. The
microscopy also confirmed the apoptosis of cancer cells on the
surface of microelectrode.144

Fig. 13 Impedance analysis as a method to examine the transition from

normal to malignant cells. The normalized resistance of the electrode,
with Rn and Rc representing the bare and cell-covered electrode resist-
ances. Growth curves for untreated control cells (NIH3T3 mouse fibro-
blasts) and cells transfected with either native (WT) or permanently acti-
vated (D143V) chemokine receptors (CXCR2) are shown. The expression
of the chemokine receptor in its native form (WT-CXCR2) resulted in a
gradual rise in resistance within 3000 seconds, followed by a stabilis-
ation at a plateau level. While, the resistance kept going up because of
the transformed chemokine receptor (D143V_CXCR2). The displayed Fig. 14 Impedance measurements to study the effect of Aspirin on HT-29
content has been replicated from ref. 138 with the authorisation of the cells. The figure reprinted from ref. 144 by permission of Taylor & Francis
publisher. Ltd; permission obtained through Copyright Clearance Center, Inc.

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Öz et al.145 employed cell-based impedance biosensors to into a host cell may result in alterations to the cellular struc-
analyse changes in the bioelectrical characteristics of NT2 cells ture. Cytopathic effects caused by viral infections, for example,
after they were treated with nucleoside analogues such as are characterised by cellular swelling, clumping, partial disin-
1β-arabinofuranosylcytosine and 29-deoxy-5-azacytidine. tegration, and, in the most extreme cases, full cellular annihil-
Unlike other approaches, such as phase contrast microscopy ation.148 Fig. 16 depicts how impedance fluctuates over time,
or gene expression profiling using PCR, they were successful in with cell attachment and spreading causing an increase in
identifying early differentiation stages during the first day of impedance and infections causing a drop in impedance.
therapy through recording alterations to impedance profiles. In addition to being able to assess the deterioration of cell
This implies that impedance sensing of the cells exhibits high monolayers in relation to pathogen loading concentration, an
sensitivity and resilience as a means of evaluating the impact added benefit of this approach is its potential to offer real-
of differentiation-inducing drugs on cellular systems. time insights into the virulence capabilities of various patho-
Consequently, they offer significant contributions to the gen strains.149 The study conducted by Fang et al.150 employed
understanding of drug mechanisms and cellular differen- mathematical modelling to analyse dynamic cellular changes
tiation processes. The electrochemical impedance biosensor obtained from electrical impedance. The findings suggest that
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could be employed to characterize the inhibition properties of the replication rate of West Nile Virus in Vero cells is approxi-
some chemicals to anti-cancer drugs. Fig. 15a shows the apop- mately three times faster than that of St. Louis encephalitis
tosis induced by treating squamous cell carcinoma cancer cell virus. An additional example of this trend is apparent in the
line (CAL 27) with different concentration of cisplatin as anti- research conducted by Nahid et al.149 wherein they investi-
cancer drug. The inhibitory effect of nicotine on anti-cancer gated the impact of four primary clinical bacterial strains on
effect of cisplatin can be seen in Fig. 15b. The results show the kinetic apoptosis of lung epithelial cells (A549). The
that any amount of nicotine reduces the effect of cisplatin on electrochemical findings indicate a restricted alteration in re-
cancer cells, with the cisplatin rendered ineffective at a nic- sistance upon Pseudomonas aeruginosa infection within a
otine concentration of 1 µM or higher.146 12-hour timeframe. However, infections with Enterococcus,
Staphylococcus aureus, and ESBL Escherichia coli all experienced
5.4 Microbiological investigation
The use of impedance-based cellular biosensors presents a
rapid and convenient approach for tracking the kinetic
response of biological cells to invading pathogens in the clini-
cal microbiology laboratory.147 The invasion of a pathogen

Fig. 16 The visual representation shows the variations in impedance

recording as time progresses. The impedance data presented in (a)
shows that the impedance of the microelectrode, measured in ohms,
Fig. 15 (a) The cell index plots of squamous cell carcinoma cancer cell undergoes a rise due to the attachment and spreading of cells. The
line (CAL 27) treated with different concentration of cisplatin as anti- exposure of infectious agents, as denoted by the arrow and markers in
cancer drug. (b) The inhibitory effect of nicotine (different concen- (b), leads to an apparent drop in impedance (ohm) due to the conse-
tration) on the apoptotic effect of 20 µM cisplatin. The displayed quent toxic effect on cells. The displayed content reproduced from ref.
content reproduced from ref. 146. Copyright 2010 Elsevier; permission 149. Copyright 2020 Elsevier; permission obtained through Copyright
obtained through Copyright Clearance Center, Inc. Clearance Center, Inc.

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a significant decline over the course of the first 5 hours, albeit advanced our grasp of cell physiology because they are so easy
with distinct patterns. to prepare in high throughput, these models have some limit-
The utilisation of impedance analysis within microbiology ations, including an inability to adequately replicate drug
laboratories for the assessment of cell/pathogen interaction penetration into tissues and the complex structure of real
has expanded beyond the determination of pathogen virulence tissue.164 The capability of an AC electrical signal to pass
capacity. McCoy et al.151 conducted a study utilising a cellular across a tissue (without interfering with or harming) makes
impedance biosensor to measure the response of MDCK cells the impedance method an attractive experimental technique
to influenza A virus. A notable finding from the research con- for screening cells in three dimensions (3D).165 Fig. 17 shows
ducted was that the administration of ammonium chloride, a the underlying concept of cellular impedance in both two- and
widely recognised viral inhibitor, resulted in an alteration in three-dimensional models of cell culture.165 In a 2D environ-
the electrical findings, suggesting that the entry of the virus ment, adherent cells are seeded directly on the surface of elec-
into the cells was impeded. Since then, cellular impedance trodes. When analysing the morphological behaviour of adher-
biosensors have been extensively employed in research endea- ent cells on microelectrodes in a 2D model, it is necessary to
vours to probe prospective innovative antiviral drugs.152–157 account for the flow of electrical signal through the resistance
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The significance of the electrical cellular biosensor in asses- of bulk medium (Rs) and the dielectric barrier of cells
sing the effectiveness of neutralising antibodies during the (Fig. 17a). By reducing the influence of solution resistance, the
2009 Influenza A (H1N1) virus pandemic was demonstrated by electrical signal provides sensitive insights about the intercel-
Tian et al.158 More recently, Charretier et al.159 proposed the lular junctions and adherence of cells to the microelectrode.
use of impedance-based cellular assays as a potential alterna- In contrast, in a 3D cell culture surroundings, cells attach to
tive to the conventional TCID50 assay for measuring viral the structural support or to other cells rather than coming into
infectious titers in vaccine development. full association with microelectrodes (Fig. 17c). When a 3D cell
Cellular impedance biosensors can potentially elucidate the culture environment is placed between electrodes, the culture
underlying mechanism of viral infections. Viruses employ media and electrodes are in direct contact with one another.
receptor binding as a mechanism to access the cellular As a result, there is always a route for current flow between
machinery of the host.160 For instance, the expression of the electrodes that escapes the cells (shunt current) which affects
AXL receptor has been widely acknowledged to facilitate the the sensitivity of measurements. By altering electrode designs,
entry of the ZIKV virus into cells.161 In order to enhance com- it is feasible to enhance the contact between cells and electro-
prehension of the mechanisms underlying Zika virus invasion des and so lower the shunt current.
of human cells, Ismail A. A. et al.162 employed cellular impe- As an example, Pan et al.166 developed a three-dimensional
dance biosensor to scrutinise the impact of viral infection on cell-based impedance biosensor (known as 3D-ECMIS) as an
brain microvascular endothelial cells. The study involved the ongoing monitoring platform for analysis of cell proliferation
use of mathematical modelling to examine impedance data. and drug efficacy testing. Two gold sensor plates were arranged
The parameters considered in the analysis included the vertically in each sensor channel of the 3D impedance bio-
barrier function of cell–cell interaction (Rb), the adhesion sensor chip, which included 8 individual culture wells on an
value of cell–surface interaction (α), and the possibility for optical base (Fig. 18). A Matrigel scaffold (Matrigel is an extra-
pathogenic agents to affect the membrane capacitance of a cellular matrix mimic) was utilized to encapsulate the human
cell. According to the ECIS results, the virus expedited the hepatoma cells (HepG2) before they were cultured in the bio-
process of vascular leakage in the brain, thereby disrupting
the permeability properties of microvascular endothelial cells
in the brain.

6. Future prospects for cellular

impedance biosensors
There are a number of emerging trends with cellular impe-
dance biosensors that are extending the scope of this powerful,
label free, method. Most notable of these is expanding the
method to 3D cell cultures and the combination of electro-
chemical impedance spectroscopy with microscopic methods
to created complementary hyphenated techniques. We will
discuss these two future trends in more detail. Fig. 17 (a) Analysing the two-dimensional cell impedance on a flat
surface. (b) Assessing the three-dimensional cell impedance when a
6.1 Three-dimensional (3D) cellular impedance biosensor scaffold-based environment is placed between electrodes. (c) Using the
scaffold-free approach to analyse the impedance of cells in three
Most frequently cells culture is performed on two dimensional, dimensions. The figure reprinted from ref. 165 with permission from
flat surfaces.163 Although cell cultures on flat surfaces have springer; permission obtained through Copyright Clearance Center, Inc.

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activities that are taking place inside of the cells.89 Despite the
many benefits of multitasking on a single platform, impe-
dance signals, due to their holistic and comprehensive nature,
are rarely able to identify particular molecular processes occur-
ring inside the cells.179 Additionally, certain significant intra-
cellular biomolecular processes have an indirect effect, or have
no influence at all, on the total impedance reading.96 An
example of this kind of concerns is the use of an electrical cell-
based biosensor as a readout to describe G protein coupled
receptor (GPCR) stimulation. The capability of cellular impe-
dance biosensors to differentiate between the Gi, Gq, and Gs
signalling pathways has been the subject of extensive
Fig. 18 (a) An impedance biosensor design for three dimensions cell research.89–91,180 Recent findings, however, have shown that
culturing. (b) Illustration of a three-dimensional impedance biosensor the cell line has a significant effect on the electrical response
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with eight channels. (c) Images of monitoring system. (d) Photographs

to GPCR activation.179 GPCRs may regulate the actin cytoskele-
taken of the platform both before and after it was seeded with live cells.
The displayed content reproduced from ref. 166. Copyright 2019 ton through a variety of proteins and second messengers.
Elsevier; permission obtained through Copyright Clearance Center, Inc. Therefore, combining label-free impedance cell-based assay as
complementary information to other techniques, especially
those that generate biologically relevant molecular insights, is
sensor device chamber. The proposed 3D-ECMIS device was a powerful approach to explain the sub-cellular processes
employed to conduct electrochemical tests demonstrating the incorporated in the impedance profile. In order to unravel the
proliferation of HepG2 cells in a Matrigel-coated 3D environ- biological processes that produce the impedance signal, our
ment. The advantages of the 3D cell culture model in cancer lab has integrated high-resolution fluorescence microscopy
research was highlighted by comparing the abilities of 3D and with cell-based impedance screening on transparent-conduc-
2D cellular impedance biosensors for anti-cancer medication tive ITO75 (Fig. 19). Evidence suggested that the reorganisation
screening (ovarian, taxol, and cisplatin). of cellular actin was apparently dependent on an increase in
There is a growing amount of research that examines the intracellular calcium. Additional research in our lab demon-
real-time analysis of cells using impedance in a three-dimen- strated, however, that the impedance approach employing ITO
sional environment,167–178 however this technique is still microelectrodes was only partially successful at gaining infor-
limited due to significant challenges. Alexander and co- mation on tiny changes in cell shape due to limited electrical
workers165 discussed the limitations in using the impedance sensitivity. Our findings181 showed that redesigning gold elec-
approach to analyse 3D in vitro systems. Briefly, there are basi- trodes and inserting a viewing window into the gold design
cally two types of 3D cell culture environments, those that use results in superior electrical sensitivity for cell impedance
scaffolds and those that do not. The possibility of toxicity or research when paired with fluorescence microscopy. Therefore,
scaffold degradation, which causes an impedance alteration, is we reported a stepwise construction method in which the gold
a significant issue when monitoring cellular impedance in micropatterns might serve as a substrate for both microscopy
scaffold-based frameworks. In addition, the scaffold has the and electrochemistry, allowing for a simultaneous dual-
potential to disrupt the effectiveness of medicine throughout sensing readout. The contribution of this biosensor to the
pharmacological therapy. Such biochemical interactions inside field is the direct visualisation of cell structures and processes
the system may play a part to the overall signal readout as a
dominant factor, hence diminishing the detection threshold.
According to Alexander et al.,165 numerous attempts have been
made to monitor cell impedance responses in scaffold-based
frameworks, but the resulting electrical output has been
mostly disappointing up until now due to the poor sensitivity
of measurement. The key benefit of the scaffold-free strategy is
that the procedure eliminates the harmful processes caused by
scaffold material and higher sensitive impedance results have
been reported.

6.2 The combination of cellular impedance biosensor with

other complementary techniques Fig. 19 The simultaneous dual sensing platform was developed to
gather more detailed data on how cells react to soluble stimuli when
In contrast to label-based assays, impedance signals are often self-assembling adhesive ligands are present on interdigitated indium tin
dependent on the identification of changes in the extracellular oxide (ITO) surfaces. The displayed content reproduced from ref. 75.
ionic environment, rather than the molecular processes and Copyright 2017 Royal Society of Chemistry.

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on the surface as high-resolution images, where microscopy

can determine the behaviour of the cells as single entities and
electrochemistry provides ensemble data on nanoscale
changes of the cells over the entire electrode.
The hyphenated approach may provide clarity on the impe-
dance data. Take for instance, the transmission of light is
dependent on the optical qualities shown by the material it
travels through, but the electrical signal is very reliant on inter-
cellular communication and cell adhesion to the surface of the
electrode. The association between the area covered by cells
and the resistance for a nonconfluent and confluent cell layer
on the electrode using simultaneous cellular impedance and Fig. 20 (a) Innovative arrangement for conducting simultaneous SPR
phase contrast microscopy analyses was shown by Choi et al.71 and cell-based impedance analysis (b and c) The effect of 5 µM cytocha-
Upon examining the two profiles, it becomes apparent that lasin D on a confluent MDCKII cell layer was measured using both SPR
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and ECIS techniques at the same time. The displayed content repro-
while time-dependent impedance findings in both profiles duced from ref. 183. Copyright 2013 Elsevier; permission obtained
indicate different stages of cell attachment on the surface, the through Copyright Clearance Center, Inc.
impedance measurements are not indicative of the surface cov-
erage. Accordingly, cells tightly adhering to a small fraction of
the electrode surface lacking intracellular attachment could
reveal the same profile as a confluent cell layer with loose beyond the detection limit of the SPR approach. This study
attachment to the surface. showed the ECIS-SPR dual sensor better characterised how
Hyphenated cell-based impedance assays have attracted cytoskeleton active medicines affect various subcellular com-
growing attention in recent years as a complement to other partments and cellular functions.
relevant cell research methods. Such hybrid techniques have Because microfluidic devices are increasingly being utilised
been used in the field, and some examples are impedance- in cell studies, the combination of microfluidics with impe-
quartz crystal microbalance (QCM),182 impedance-surface dance-based measurements of cells is becoming increasingly
plasmon resonance spectroscopy (SPR),183 impedance-light- popular.185–189 Impedance implementation in microfluidic
addressable potentiometric sensor (LAPS)184 and impedance systems may be useful from a number of perspectives.
flow cytometry.185 More precise data may be generated with Integrating impedance microelectrodes into microfluidic
the help of cell-based hybrid biosensors. Impedance and devices enables detailed analysis of single cells rather than cell
surface plasmon resonance (SPR) cell-based assays are two populations opening the door to more fundamental biological
such approaches that, when used independently, show the research.189 Additionally, impedance flow cytometry does not
advantages and disadvantages of each method for answering need adherent cells. The fact that a majority of cell lines
certain questions. exhibit a lack of adherence to gold or glass electrodes presents
The optical SPR readout only identifies changes near to the a technical hurdle in classical cell–substrate impedance
substrate surface (200 nm). Tight junction which allows cells measurement. Controlling cell adhesion prior to doing the
to communicate with other cells is an example of a morpho- experiment using a traditional impedance technique is there-
logical change that lies above the evanescent wave of SPR. The fore crucial, and the fabrication of bio-interfaces or an artifi-
electrical impedance readout, however, gives information cial extracellular matrix may be necessary. These bio-interface
about the entirety of the cell body, encompassing interactions materials, however, could not precisely resemble the extracellu-
between cells and between cells and substrates. Therefore, lar matrix, which might change the shape and adhesion
surface plasmon resonance (SPR) integration on an impedance characteristics of cells.58
cell-based platform has the potential to enhance comprehen- Ayliffe and colleagues190 were the pioneers in developing a
sion of morphological alternations occurring at the subcellular microfluidic system that incorporated microelectrodes for the
level. Using the developed dual biosensor, Michaelis et al.183 purpose of detecting cellular bioelectrical activity at the single
employed confluent MDCK II cells to investigate the impact of level. Single cell electrical characteristics may serve as biologi-
cytochalasin D (CD). To evaluate the cellular response to cyto- cal biomarkers to categorise different type of cells including
skeletal rearrangement, 5 µM CD was introduced to MDCK cell blood cells, tumour and stem cells.191 Microfluidic impedance
monolayer while SPR and impedance experiments were con- cytometry outcome is based on the biophysical aspects of cells
ducted simultaneously. SPR and impedance time courses like their size, shape and the dielectric properties of their
show that the decrease in impedance exhibits a more rapid membranes. Numerous studies have shown the capacity of
rate of change in comparison to the decline in reflectivity impedance cytometry systems to distinguish cancer cells utilis-
(Fig. 20). The SPR readings predominantly disclose the cytos- ing various designs.192–197 One example is a microfluidic impe-
keleton transformation near the lower cell body, but impe- dimetric device designed to identify leukaemia cells. It was
dance measurements (at low frequencies) are indeed very sen- demonstrated that normal red blood cells (RBCs) and cancer-
sitive to alternations in the junctions of cell–cell, which is ous ones could be effectively differentiated.198 The signal to

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noise ratio (SNR) was examined by altering the excitation demonstrating strong metastatic potential. The findings of the
voltage and ionic concentration in solution. The channel geo- impedance recording showed that the phase component of
metry was also optimised to enhance the sensitivity of the impedance may be utilised to discriminate between the meta-
output signal. Human red blood cells (RBCs) and leukaemia static condition of HNC cells at the level of single-cell.
cells (LCs) were separately suspended in a 2.5 S m−1 conduct- In another study, Nguyen et al.201 developed an impedance-
ing buffer solution. By pumping suspensions to the channel at integrated microfluidic system utilising the Boyden chamber
a pressure difference of 30 mbar and a release rate of five concept, which is comprised of three primary components: a
microliters per hour, a flow of 2000 cells per min was attained. microfluidic channel, cell capture spots, and an impedance
When a voltage of 3 V was applied to the electrode and a microelectrode. The device enabled to rapidly and selectively
1.5 MHz electric pulse was generated, both cell types produced identify migratory characteristics of single cancer cells. More
distinct signal waveforms with a focus on the single events recently, a microfluidic device was developed202 allowing sim-
(Fig. 21). ultaneous investigation of static and dynamic cells by combin-
A single cell may be precisely contained at a specific ing electric impedance flow cytometry and EIS on the same
location of microfluidic structural using innovative designs, platform (Fig. 22). The tool has been shown to be effective at
Published on 23 November 2023. Downloaded on 12/2/2023 4:07:44 AM.

opening novel possibilities for combinations with impedance both letting dynamic cells through for impedance flow cytome-
microelectrodes. According to the published literature, there try assessments and capturing them for EIS readings. The
are two categories of impedance biosensors that use microflui- entire impedance spectrum may be recorded by EIS, which
dic technology.199 Microfluidic impedance cytometry may be provides a broad range of data about the unique properties of
used to analyse either dynamic or static cells (that have been the cells. HepG2, HeLa and A549 cancer cell populations were
trapped in a microcavity structure). As an example of a station- separately assessed using the microfluidic device. Impedance
ary cell examined through a spectrum of impedance utilising a flow cytometry readout revealed that the magnitude degree of
microfluidic device, Cho et al.,200 combined flow channel for impedance was very different between malignant cells. While
cells with cell trapping sites and opposing microelectrode the whole impedance spectrum of a single cell, which was
arrays for impedance measurement. By exerting suction via the recorded by EIS, was employed to obtain specific biophysical
microchannel designed specifically for cell trapping, a single information about each cell type, including the dimension of
cell was pulled into the microfluidic cell capture site as it the cells, specific cellular capacitance, and intracellular resis-
moved down the cell flow channel. Upon capturing an individ- tivity. This work showed that integrating EIS to impedance
ual cell within the designated electrical analysis spot, the flow cytometry presents a novel method to expand knowledge
syringe pump was promptly turned off. Following this, impe- regarding electrical characteristics of single cells.
dance analysis was performed on the captured cells using a More than 50 years of research have focused on the corre-
voltage of 500 mV, spanning a frequency spectrum of 40 Hz to lation of cell deformability and chronic illnesses.203
10 MHz. The study acquired impedance spectrum data from Consequently, the deformability of cells may serve as a funda-
two different head and neck carcinoma (HNC) cell lines, mental biomarker for diagnosing diseases.203 The biomechani-
characterised by varying degrees of metastatic potential, with cal characteristics of a cell can be measured by deforming the
one exhibiting limited metastatic potential and the other cell. One effective way to induce mechanical stimuli is using
constriction channels, which are only slightly smaller in dia-
meter than the measured cell sizes.204 When cells are pushed

Fig. 22 (a) The schematic representation of the microstructure com-

Fig. 21 Histogram plots and peak amplitude for (a) red blood cells and bining electrical impedance spectroscopy and impedance flow cytome-
(b) white blood cells based on the impedance flow cytometry signals for try shows the cell flowing or trapping. (b) The variability of flow velocity
single particle events. The displayed content reproduced from ref. 198. within the channels from an overhead perspective. Reproduced from
Copyright 2020 Elsevier; permission obtained through Copyright ref. 202. Copyright 2019 American Chemical Society; permission
Clearance Center, Inc. obtained through Copyright Clearance Center, Inc.

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 View Article Online

Critical Review Analyst

dance approach has promising analytical applications for ana-

lysing adherent cells. In contrast to label-based assays, this
non-invasive approach eliminates the necessity of labelling
and offers superior temporal resolution, enabling real-time
surveillance of native cell populations over timeframes of
seconds to days.
While traditional impedance cell-based assays offer advan-
tages in terms of multitasking on a single platform, they face
challenges when unravelling fundamental biological ques-
tions. Some of these challenges are common to all cell-based
biosensors, such as the fact that real samples have a diverse
cell population. Other challenges, not limited to singular bio-
Fig. 23 A microfluidics to examine the deformability of cells together chemical events, are specific to the nature of the impedance
with an associated impedance analysis. Reproduced from ref. 204. signal. Although there are some gaps and limitations of estab-
Published on 23 November 2023. Downloaded on 12/2/2023 4:07:44 AM.

Copyright 2012 American Chemical Society; permission obtained

through Copyright Clearance Center, Inc.
lished commercial impedance assays including ECIS,
xCELLigence and Cell Key,58,71,75,96,183,205 impedance cell-
based assays are likely to become more widespread over the
coming years due to their ability to monitor native cell popu-
into a constriction channel, they are compressed by the narrow
lations in real time.
walls. A number of characteristics related to cell deformability
To enhance measurement and help in the interpretation of
may be measured, including the time of transit and cell
complicated cellular processes during cell monitoring, a multi-
stiffness. A microfluidic device that integrates impedance ana-
faceted strategy is used that integrates different approaches
lysis with a constriction channel was developed by Adamo and
with impedance analysis. Hyphenated strategies, such as impe-
colleagues204 (Fig. 23). It was shown that larger cells had sig-
dance-SPR, have been shown to be beneficial for identifying
nificantly slower travel times than smaller ones. To verify that
specific alterations in cell morphology at the subcellular level.
cell stiffness affects transmit time, actin-disrupting agents
Additionally, combining high-resolution fluorescence
(latrunculin A and cytochalasin B) were introduced to HeLa
microscopy with cell-based impedance screening can provide
cells. The untreated cells (the population that was stiffer) took
invaluable insights into complex biological processes that
longer to pass through the constriction channel (narrowing in
influence cell dynamics.
the form of a funnel) indicating that the medicine affected the
Furthermore, to overcome challenges caused on by the con-
actin filament. The core of this label-free integrated platform
voluted nature of impedance signals, future endeavours may
for data generation is the combination of mechanical and elec-
concentrate on improving the adaptability of technique to a
trical attributes of the cells simultaneously. Peak widths
particular biological process. Finding novel strategies to
exhibit travel time, which is impacted by mechanical pro-
enhance the sensitivity and specificity of cellular impedance
perties of the cell, while impedance spikes reflect cellular elec-
analysis will allow for more informative data.
trical characteristics. Cell size affects these two biomarkers in
Another future direction for development is investigating
different ways. Ghassemi et al.205 were the first researchers to
cell impedance measurement in three-dimensional (3D) cellu-
employ this specific integrated approach as alternative strategy
lar settings. Drug assessment and advancement might be
to instantaneously count circulating tumor cells in blood
improved by constructing 3D cellular impedance biosensors,
samples within a minute, overcoming the necessity for fluo-
notwithstanding the challenging nature of this endeavour.
rescent tagging. The key concern with circulating tumor cells
Examining single cells owing to the diverse nature of actual
examination technologies is that there are only a few circulat-
cell samples, eliminating the adherent cells requirement and
ing tumor cells per millilitre of blood, while there are billions
minimizing the duration for preparation and testing are all
of other blood cells all around them. Normal blood cells often
crucial steps towards improving the therapeutic relevance of
have a smaller size than circulating tumour cells, which results
impedance technique. Impedance-based microfluidic devices
in distinct impedance profiles when cancer cells undergo
have the potential for clinical applications by simplifying the
transit related deformation while normal blood cells maintain
procedure and providing instantaneous screening of individual
their original shape.
cells under minutes.
In conclusion, there is a great deal of room for growth and
improvement in the area of impedance analysis of cells, which
7. Conclusion is itself quickly expanding. To further improve this approach
and open up new avenues for understanding cell behaviour in
Electrochemical impedance for monitoring cells has quickly a wide range of biological contexts, researchers can focus on
gained popularity as a relevant screening process across areas such as integrating it with other complementary
different biological disciplines due to its flexibility and simpli- approaches, enhancing its specificity, examining 3D settings,
city of use. The literature suggests that the traditional impe- and optimising its integration with microfluidics. With persist-

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Analyst Critical Review

ent efforts and creative ideas, impedance monitoring of cells is 14 P. Jin, Z. Ren, F. Ye and W. Ying, Anal. Biochem., 2014,
prepared to significantly advance biological study and health 450, 27–29.
care applications. 15 T. Söllradl, F. A. Banville, U. Fröhlich, M. Canva,
P. G. Charette and M. Grandbois, Biosens. Bioelectron.,
2018, 100, 429–436.
Author contributions 16 J. S. Daniels and N. Pourmand, Electroanalysis, 2007, 19,
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Seyedyousef Arman: conceptualization, writing – original draft, 17 R. Halai, D. E. Croker, J. Y. Suen, D. P. Fairlie and
writing – review & editing. J. Justin Gooding and Richard M. A. Cooper, Biosensors, 2012, 2, 273–290.
D. Tilley: supervision, conceptualization, funding acquisition, 18 B. Liedberg, C. Nylander and I. Lundström, Biosens.
writing – original draft, writing – review & editing. Bioelectron., 1995, 10, i–ix.
19 M. Hide, T. Tsutsui, H. Sato, T. Nishimura, K. Morimoto,
S. Yamamoto and K. Yoshizato, Anal. Biochem., 2002, 302,
Conflicts of interest 28–37.
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20 V. Chabot, C. M. Cuerrier, E. Escher, V. Aimez,

There are no conflicts to declare. M. Grandbois and P. G. Charette, Biosens. Bioelectron.,
2009, 24, 1667–1673.
21 N. Zaytseva, W. Miller, V. Goral, J. Hepburn and Y. Fang,
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22 Y. Fang, A. M. Ferrie, N. H. Fontaine, J. Mauro and
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the National Health and Medical Research Council Project 23 A. M. Ferrie, Q. Wu and Y. Fang, Appl. Phys. Lett., 2010, 97,
grant (GNT11662385) and an NHMRC Investigator grant 223704.
(GNT1196648). S. A. also acknowledges the Australian Federal 24 Q. Yang, X. Huang, B. Gao, L. Gao, F. Yu and F. Wang,
Government for funding under the Research Training Analyst, 2023, 148, 9–25.
Program. 25 B. Markhali, R. Naderi, M. Mahdavian, M. Sayebani and
S. Arman, Corros. Sci., 2013, 75, 269–279.
26 U. Tröltzsch, O. Kanoun and H.-R. Tränkler, Electrochim.
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