Abstract
The use of electrochemical methods to study living systems, including
cells, has been of interest to researchers for a long time. Thus,
controlling the polarization of the electrode contacting living cells,
one can influence, for example, their proliferation or the synthesis of
specific proteins. Moreover, the electrochemical approach formed the
basis of the biocompatibility improvement of the materials contacting
with body tissues that use in carbon hemosorbents and implants
development. It became possible to reach a fundamentally new level in
the study of cell activity with the introduction of optically
transparent electrodes in this area. The use of such materials allowed
approaching to the study of the influence of the electrode potential on
adhesion activity and morphology of the different cell types (HeLa
cells, endothelial cell, etc.) more detailed. There are a negligible
number of publications in this area despite the obvious advantages of
the usage of optically transparent electrodes to study living cells.
This mini review is devoted to some aspects of the interaction of living
cells with conductive materials and current advances in the use of
optically transparent electrodes for the study of living cells, as well
as the prospects for their use in cellular technologies.
Keywords: living cell, optically transparent electrode, indium
tin oxide, polarization, morphology, cell adhesion
1. Introduction
Interest in using electrochemical methods to study living systems has
been evident for decades. The simplicity, selectivity, sensitivity and
relatively low cost of electrochemical methods allowed them to be widely
introduced in the field of medicine and biology. Moreover, their use is
not limited only to analytical applications, but also underlies a number
of medical procedures (electrochemical hemostasis, electrochemical
lysis, iontophoresis, indirect electrochemical detoxification,
application biocompatible coatings for endoprostheses and others
(Sawyer, Srinivasan, Stanczewski, Ramasamy & Ramsey, 1974, Nilsson et
al., 2000, Djokić, 2016, Gratieri, Santer & Kalia, 2017)).
2. Prerequisites of studies of the interaction of living cells with
electrically conductive materials
Since the 50s of the last century, active work is being done to study
the interaction of cells with foreign charged materials. For the first
time, the assumption that the surface charge of an electrically
conductive material affects the interaction of blood cells with this
material was put forward when studying the mechanism of thrombogenesis.
When blood contacted with metals having a positive potential value, a
thrombus formed, while blood contacted with metals having negative
potential values did not produce a blood clot. It was found in
vivo that the value of the electrode potential of the order of +0.5 V
(Ag/AgCl) was a critical value, after which there was an increased
thrombus formation at the site of contact of blood and metal (Sawyer,
Srinivasan, Stanczewski, Ramasamy & Ramsey, 1974). These data were
consistent with in vitro experiments with external polarization,
where it was shown that red blood cells and white blood cells were
deposited on the surface of platinum and gold electrodes at a potential
of +0.55 V (Ag/AgCl) (Saywer, Brattain & Boddy, 1964) and platelets
were deposited at a potential of +0.65 V (Ag/AgCl) (Sawyer, Srinivasan,
Stanczewski, Ramasamy & Ramsey, 1974). The phenomenon of cell
deposition on the electrode was associated by the authors with the
transfer of part of the charge from the cell membrane to the electrode
surface, i.e. with the course of the electrochemical process. Red blood
cell adhesion was also studied on a lead electrode in a solution of
0.012 M NaF at various electrode charge densities (Gingell & Fornes,
1976).
In connection with the development of technologies in tissue
engineering, there is an increase in interest in research in this area,
as evidenced by studies on the electrochemical adhesion and desorption
of various types of cells (Jiang, Ferrigno, Mrksich & Whitesides, 2003,
Inaba, Khademhosseini, Suzuki & Fukuda, 2009, Sun, Jiang & Jiang,
2011, Enomoto et al., 2016, Kobayashi et al., 2019).
Electrochemical ideas about the interaction of blood cells with a
charged surface were used to study the traumatic ability of hemosorbents
based on activated carbons in relation to blood cells (Goldin, Volkov,
Goldfarb & Goldin Michael, 2006). Thus, the possibility of reducing the
traumatic effect by external polarization of the hemosorbent was shown,
and a potential region from -0.15 to +0.05 V (Ag/AgCl) was revealed, in
which activated carbon remained indifferent to blood cells. The results
formed the basis for the creation of composites based on activated
carbon with an electrically conductive polymer deposited on its surface,
which made it possible to create hemosorbents with desired properties
without the need for external polarization (Khubutiya, Goldin, Stepanov,
Kolesnikov & Kruglikov, 2012, Volfkovich, Goroncharovskaya, Evseev,
Sosenkin & Goldin, 2017).
In addition, with the help of external polarization it is possible to
regulate cell proliferation and cell synthesis of specific proteins. For
example, under conditions of external polarization of the platinum
electrode when the potential is shifted from +0.1 V (Ag/AgCl) to the
region of more positive potentials, a decrease in the proliferation
(division) of MKN45 line cells is observed, and at +0.4 V (Ag/AgCl) its
complete inhibition (Kojima et al., 1991), and at the potential of about
+0.4 V (Ag/AgCl), the maximum yield of carcinoembryonic antigen (CEA)
synthesized by these cells is observed (Kojima et al., 1992).
Recently, studies of the electrochemical behavior of blood cells have
resumed, which have experimentally proved the existence of electron
transfer between cells and the electrode surface. Thus, it was found
that erythrocytes undergo electrochemical reduction at a platinum
electrode at potentials less than -0.15 V (Ag/AgCl), and electrochemical
oxidation at potentials more positive than +0.2 V (Ag/AgCl) (Tsivadze et
al., 2017).
The phenomena described above took place upon contact of cells with a
foreign electrically conductive material under conditions of external
polarization. At the same time, when studying a number of bacterial
cells (for example, E. Coli , Geobacter sulfurreducens ,
etc.), the opposite effect of unidirectional transport of electrons
generated during the course of enzymatic processes from cell to
electrode was discovered, which formed the basis for the development and
creation microbial fuel cells (Bond & Lovley, 2003, Marsili et al.,
2008, Scott & Yu, 2015).
3. Optically transparent electrodes
The introduction of optically transparent electrodes (Ellmer, 2012) used
mainly in the production of liquid crystal displays and solar panels,
has allowed reaching a qualitatively new level in studies of the
interaction of living cells with electrically conductive materials. A
wide range of materials can be used as optically transparent electrodes,
from thin metal layers (for example, silver and copper widely used in
the electronic industry (Bi et al., 2019) to the most innovative
materials (for example, carbon nanotubes (López-Naranjo, González-Ortiz,
Apátiga, Rivera-Muñoz & Manzano-Ramírez, 2016), electrically conductive
polymers (3,4-ethylenedioxythiophene) (PEDOT) (Hofmann, Cloutet &
Hadziioannou, 2018) and graphene oxide (Woo, 2019)) deposited on a
transparent substrate. However, the most widely used are optically
transparent electrodes based on indium and tin oxides (ITO) (Cao, Li,
Chen & Xue, 2014, Afre, Sharma, Sharon & Sharo, 2018), which have a
fairly high light transmission (80–95%) with a relatively low
resistance (10–50 Ω/cm2) (Cao, Li, Chen & Xue, 2014).
4. The use of optically transparent electrodes for the study of living
cells
Despite the obvious advantages when using optically transparent
electrodes for the study of living cells, namely, the ability to
visualize the behavior of cells in conditions of external polarization,
this property was not realized in early works. In this case, either the
effect of external polarization was studied without using the optical
properties of the material, or the behavior of cells was visually
examined, but without external polarization.
So, on the one hand, under conditions of external polarization of the
ITO electrode without visual control, when studying mouse astrocyte
cells, it was shown that at a potential of +0.3 V (Ag/AgCl) maximum
secretion of neuron growth factor (NGF) is observed (Koyama, Haruyama,
Kobatake & Aizawa, 1997) when studying thrombus formation, it was
revealed that cathodic polarization prevented the formation of a
thrombus (Schmitt, Baer, Meindl, Anderson & Mihm, 1984).
On the other hand, optically transparent electrodes can be used as the
basis for creating cell patterns. For example, when a part of the ITO
electrode surface is coated with gold modified to give it resistance to
cells, the cells will selectively adhere to the ITO surface, which was
controlled by the optical properties of the material (Jin, Yang, Zhang,
Lin, Cui & Tang, 2009).
From our point of view, it is important to use the optical properties of
materials to study their interaction with blood cells. Thus, in the
absence of external polarization, it was shown that when red blood cells
come in contact with materials such as glass coated with
polydimethylsiloxane (-24 mV), glass coated with ITO (-39 mV) and glass
without coating (-60 mV), the red blood cell morphology is shifted from
discocytes to echinocytes as the surface potential shifts to more
negative values (Mukhopadhyay, Ghosh, Sarkar & DasGupta, 2018).
From the electrochemical point of view, the phenomena of changes in cell
morphology can be explained by changes in the transmembrane potential.
For example, it is known that a change in the morphology of red blood
cells, i.e. the transition stomatocyte ↔ discocyte ↔ echinocyte affects
the change in the double electric layer at the erythrocyte membrane due
to the influence of ionic strength and osmolarity of the environment
(Bifano, Novak & Freedman, 1984, Glaser, 1993). Moreover, it was shown
(Tachev, Danov & Kralchevsky, 2004) that this mechanism does not
contradict the widespread bilayer pair theory (Sheets & Singer, 1974),
where, as is believed, the band 3 protein plays the main role in the
mechanism of changing the shape of the red blood cell (Betz, Bakowsky,
Müller, Lehr & Bernhardt , 2007).
In connection with the foregoing, an obvious conclusion is drawn about
the prospects of studying the interaction of living cells with optically
transparent electrodes in conditions of external polarization.
Schematically, the possibilities of using optically transparent
electrodes for studying cells are shown in Figure 1.
The most detailed studies of the influence of the potential of an
optically transparent electrode on cell morphology were performed using
cultures such as HeLa cells, bovine aortic endothelial cells, P.
Fluorescens , etc. (Yaoita, Shinohara, Aizawa, Hayakawa, Yamashita &
Ikariyama, 1988, Wong, Langer & Ingber, 1994, Busalmen & de Sánchez,
2005).
When studying the effect of the ITO electrode potential on the
morphology and growth of living HeLa cells at ITO potentials below +0.5
V (Ag/AgCl) no changes were observed, in the potential range from +0.5
(Ag/AgCl) to +0.7 V (Ag/AgCl) there is a reversible change in cell
morphology and a decrease in cell proliferation rate, and at potentials
above +0.7 V (Ag/AgCl) irreversible morphological changes begin, leading
to cell death (Yaoita, Shinohara, Aizawa, Hayakawa, Yamashita &
Ikariyama, 1988, Yaoita, Aizawa & Ikariyama, 1989, Yaoita, Ikariyama &
Aizawa, 1990).
Also, using HeLa cells as an example, their adhesion and desorption from
the surface of an ITO electrode depending on the applied potential was
shown (Koyama, 2011). HeLa cells adhered to the ITO surface at +0.4 V
(Ag/AgCl), while at -0.3 V (Ag/AgCl), the cells adhered to areas of
glass not coated with ITO.
The behavior of bovine aortic endothelial cells on an ITO electrode
coated with polypyrrole was studied by Wong, Langer & Ingber (1994).
The authors note that on an oxidized polypyrrole film (without imposing
potential), the cells attach to the surface and are flattened, at the
same time, when the film is brought into a neutral state (-0.5 V), the
cells attach, but remain round.
Studies on gold coated glass of P. Fluorescens culture (Busalmen
& de Sánchez, 2005) showed that at a potential of -0.5 V (Ag/AgCl),
bacteria do not adhere directly to the electrode surface, but form
clusters, in contrast to the potential range from 0.1 In (Ag/AgCl) to
-0.2 V (Ag/AgCl), where the cells showed exponential growth with a
doubling time of 82.6 ± 7 min. At the same time, two different effects
were detected in the anodic potential region. So, at +0.5 V (Ag/AgCl) an
exponential growth is observed with a doubling time of 103 ± 8 min,
while at +0.8 V (Ag/AgCl) the bacteria did not grow and a decrease in
the area covered by bacteria was observed over time their entrainment
from the surface with a solution.
In a study on an ITO electrode and an electrode based on gallium zinc
oxide doped with yeast cells of S. Cerevisiae strain, it was
shown that the cells attach to the electrode and show normal
proliferation on the ITO electrode in the potential range from -0.2 V to
-0.4 V (Ag/AgCl), while cell attachment to gallium doped zinc oxide did
not occur (Koyama et al., 2015).
Regarding blood cells, as the most relevant object for research, it
should be noted (Tsivadze et al., 2017, Goldin, Goroncharovskaya,
Evseev, Shabanov, Goldin & Petrikov, 2019), where it was shown that the
morphological state of red blood cells depends on the potential of the
ITO electrode. In potentiodynamic conditions, i.e. with a linear sweep
of the potential into the cathodic and anodic potential regions, the
initial normal red blood cells (discocytes) were transformed into
various morphological forms (echinocytes, spherocytes, stomatocytes)
(Figure 2).
It is important that each of the morphological forms is formed and
exists in certain ranges of potentials. When the ITO electrode potential
shifts to the cathodic potential region at potentials less than -0.25 V
(Ag/AgCl), transformation of discocytes into echinocytes was observed,
which, when the potential shifts to even more negative values,
transforms further into spheroechinocytes. When the ITO electrode
potential shifted to the anodic potential region to a potential of +0.6
V (Ag/AgCl), no morphological changes were observed; more positively,
the transformation of discocytes into stomatocytes took place. It is
also important that the observed transformation of red blood cells was,
as a rule, reversible. So, when changing the direction of scanning the
potential, or turning off the current, a transition of the red blood
cell to the initial state was observed.
The use of electrochemical impedance spectroscopy using optically
transparent electrodes allows one to obtain additional information on
the functioning of cells, for example, the synthesis of biologically
active substances, the activity of ion channels, and adhesive activity
(Choi, English, Jun, Kihm & Rack, 2007, Choi, English, Kihm &
Margraves, 2007, Jahnke et al., 2009, Pänke, Weigel, Schmidt, Steude &
Robitzki, 2011, Jahnke, Schmidt, Frank, Weigel, Prönnecke & Robitzki,
2019).
5. Conclusions
The data presented in this mini review indicate the prospects of
developments in the field of application of optically transparent
electrodes for the study of living cells. Although questions remain
about the nature of the observed phenomena in the blood cell / foreign
charged surface system, the basis for answering these questions has
already been laid. Obviously, the topic raised is highly relevant due to
its interdisciplinarity, since it touches on a wide range of problems
from identifying new fundamental aspects of the interaction of cells
with charged surfaces to the application of optically transparent
electrodes in cell engineering. In general, conducting research in this
area using modern methods of analysis should not only solve fundamental
problems, but also serve as a basis for the development of new
diagnostic and therapeutic methods.