Elsevier

Biosensors and Bioelectronics

Volume 216, 15 November 2022, 114621
Biosensors and Bioelectronics

Scanning electrochemical microscopy based irreversible destruction of living cells

https://doi.org/10.1016/j.bios.2022.114621Get rights and content
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Abstract

In this research, scanning electrochemical microscopy combined with electrochemical impedance spectroscopy has been applied to irreversible electroporation of active yeast cells by causing cell death. This finding is important for the development of irreversible electroporation technique, which could be suitable for the curing of cancerous tissues, because during this research cell death has been achieved using relatively low ultramicro-electrode (UME) voltage, precisely of 2.0 V vs Ag/AgCl,Cl-sat. It was determined that the irreversibly electroporated area of immobilized yeast cells was located directly below the UME and was of approximately 20 times larger width than the diameter of the UME, leaving undamaged cells out of this area. The ability of SECM to move the UME with high accuracy in x, y, and z directions and the ability to use electrodes of various diameters as well as the fact that the diameter of the electroporated area depends on the diameter of the UME and on the distance between the UME and the surface, what offers the possibility to establish targeted electroporation systems for selective treatment of tissues.

Keywords

Electrochemical tissue removal
Irreversible electroporation
Yeast cells
Scanning electrochemical microscope
Electrochemical impedance spectroscopy
Ultramicro-electrode

1. Introduction

Each living cell is surrounded by a plasma membrane that controls the exchange of substances and is involved in the transmission of signals from the environment (le Roux et al., 2019). In 1925 Gorter and Grendel showed that the membrane is composed of two layers of self-organized phospholipid molecules(Gorter and Grendel, 1925). These are amphiphilic molecules having a hydrophilic head consisting of a phosphate group and glycerol and a hydrophobic tail consisting of two long fatty acid chains connected via ester linkages to the first and second carbon atoms of the glycerol chain (Suetsugu et al., 2014). The membrane is stabilized by hydrophobic effects and van der Waals interactions. In an aqueous solution, the hydrophobic tails of phospholipids begin to self-organize in order to reduce interaction with water molecules and spontaneously form closed structures composed of a double layer based on lipid molecules. The polar heads face the water, and the hydrophobic tails is directed in the inside of the bilayer (Andersson Trojer and Brezesinski, 2016; Marsh, 2016).
Cell membranes possess selective permeability that can be modified and one of the modification methods is electroporation. It is a physical method in which cells are exposed to a strong electrical pulse (about 1 kV cm−1), which results in the formation of temporary or permanent pores in the phospholipid layer (Kotnik et al., 2019). The first reports of transient permeability changes in vesicle membranes as a result of exposure to an external electric field were published in 1972. The procedure was called electroporation, and it was divided into two main branches: reversible electroporation, in which the treated cells have restored their initial state and survived, and irreversible electroporation, which resulted in cell death (Neumann and Rosenheck, 1972).
When cells are exposed to an electric field, a transmembrane potential (ΔΨm) is induced in the plasma membrane of the cells, the magnitude of which depends on the strength of the external field and the radius of the cell, which can be defined with the formula (1):(1)ΔΨm = 1.5 Eext R cosθWhere Eext is the strength of the external electric field; R – cell radius; cosθ – indicates the angular position of the cell with respect to the electric field vector.
A membrane is electroporated when its potential reaches a critical threshold from 200 mV up to 2.0 V, which is depending on the pulse duration (Batista Napotnik and Miklavčič, 2018; Delemotte and Tarek, 2012; Venslauskas and Šatkauskas, 2015).
Electroporation is a probabilistic process, therefore, the number and size of pores in the membrane is highly dependent on the basic physical parameters: pulse potential, pulse shape, pulse duration, and number of pulses (Bodénès et al., 2019; Demiryurek et al., 2015). High-potential long-term electrical impulses can completely collapse the integrity of the cell membrane because such pulses induce expanding pores beyond the critical size. The destruction of cell membrane causes its death (Bodénès et al., 2019; Thomson et al., 2015). Irreversible electroporation for quite a long time has been interpreted as a side effect and has only been investigated by setting cut-off values of the electric field at which cells remain viable. However, irreversible electroporation can be applied to the food industry – food and liquid sterilization. Up to 99.99% of bacteria are killed by a short pulse of a strong electric field when the bacterial suspension is exposed. The basis of such sterilization is the damage of cell membranes by a strong electric field (Kotnik et al., 2015; Saito et al., 2013). Research over the past few years has shown that irreversible electroporation can become an important and promising treatment method in recent biomedicine (Cohen et al., 2018; Geboers et al., 2020; Paiella et al., 2016). It has been found that the application of irreversible electroporation can successfully remove cancerous tissue or tumor without damaging healthy tissues. One of its most important advantages is that no drugs are used. In this case, one of the main aspects is the ability to precisely control the size of the electroporated area (Paiella et al., 2015; Wagstaff et al., 2016).
Scanning electrochemical microscope (SECM) can be applied for the investigation of living cells (Morkvenaite-Vilkonciene et al., 2016; Petroniene et al., 2020a, 2020b; Ramanavicius et al., 2017) and was applied the key tool for the development of a targeted cell-electroporation system (Poderyte et al., 2022). SECM is a useful tool for the investigation of complex and sensitive biological systems since while measuring, the analyte is not damaged in any way (Polcari et al., 2016). SECM signal is a current that is measured by ultramicro-electrode (UME), this current is induced by oxidation/reduction reaction that took place on the working surface of UME. The UME size can vary from the diameter of 25 μm down to a few nanometers and it can be moved in all three x-, y-, z-dimensions with the accuracy of 1 μm (Izquierdo et al., 2018; Zoski, 2016). It was already demonstrated in our previous research that SECM can be applied to electroporate living yeast cells (Poderyte et al., 2022) and artificial phospholipid bilayer membranes (Valiūnienė et al., 2020a). It has been shown that the size of electroporated area by SECM depends upon applied electrode diameter (Danieli and Mandler, 2013; Grisotto et al., 2011). The same correlation can also be seen during the application of SECM for the electroporation (Poderyte et al., 2022; Valiūnienė et al., 2020a).
In the light of these findings in this research, a scanning electrochemical microscope was successfully adapted to study the electroporation process of living yeast cells (Fig. 1). To the best of our knowledge, the applicability of SECM for electroporation process performance and its investigation has never been published by any other research groups excluding our group previous work (Poderyte et al., 2022). Considering that SECM is highlighted as a not invasive and not destructible technology, which can be applied for the assessment of sensitive biological systems (Morkvenaite-Vilkonciene et al., 2019). As we showed in our recent work (Poderyte et al., 2022) fully reversible electroporation can be obtained while only using SECM-based technology. Meanwhile, in this research we have shown that by the adjustment of proper system parameters, this SECM-based technology can also be applied for irreversible electroporation as well. The ability of SECM to move the UME in the x, y, and z directions with high accuracy, the ability to use electrodes of different diameters, and the dependence of the size of the formed pores on the diameter of the electrode used for the electroporation – has been adjusted in this research to create controlled irreversible electroporation model. The novelty of this research is the applicability of SECM to perform the electroporation process using a three-electrode system using relatively low voltage (2.0 V) pulses and to damage the cells only directly below the working electrode, leaving untouched these cells, which are out of this area. All these findings could lead to a breakthrough in developing targeted irreversible electroporation based treatment of cancerous tissue removal (see Fig. 2).
Fig. 1
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Fig. 1. Visualization of possible electroporation process by voltage of 2.0 V applied for 120 s to the Ø 25 μm UME, which is positioned at 20 μm distance above the surface of active yeast cells.

Fig. 2
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Fig. 2. Cyclic voltammetry curve of the ⌀ 25 μm Pt ultramicro-electrode in buffer solution, pH 7.1, with 10 mM of glucose. Potential scan rate 10 mV s−1.

2. Experimental part

2.1. Ultramicro-electrode preparation

Before starting electrochemical measurement, ultramicro-electrode was cleaned carefully, since it is a very sensitive system, in which any impurities could cause contamination of the working area. The platinum UME of 25 μm diameter from Sensolytics (Bochum, Germany) was cleaned both mechanically and electrochemically. First of all, the electrode was rinsed with ethanol and then mechanically polished with sandpaper with a grain size of 0.3 μm while scrubbing it in a circular motion and holding it perpendicular to the sanding surface. After polishing, the electrode was washed with distilled water and placed in a three-electrode cell with 0.5 M H2SO4 solution where a Pt wire was used as the counter electrode, Ag/AgCl,Cl-sat – as the reference electrode, and the UME – as the working electrode. To wash the electrode electrochemically, cyclic voltamperograms were recorded in the potential range from 0 V to 1.7 V at a scan rate of 0.2 V s-1 (10–50 cycles) until no undesired redox peaks occur.
The same three-electrode cell was used to determine the electrode potential suitable for further electrochemical measurements at potentiostatic mode. The cleaned UME was placed in an electrochemical cell far from the surface of interest (at least 1000 μm away) and cyclic voltamperograms were recorded in a 0.01 M phosphate-buffer, pH 7.1, in the potential range from −0.9 V to 0.9 V vs Ag/AgCl,Cl-sat. The phosphate-buffered saline (PBS) solution was prepared by dissolving 0.1 M NaCl and 0.01 M NaH2PO4•2H2O in distilled water. Then it was adjusted with NaOH solution until a pH of 7.1 was reached.

2.2. Preparation and immobilization of active and inactivated yeast cells

The active yeast suspension has been made by adding 50 mg of dry yeast (Saccharomyces cerevisiae) and 0.5 ml of deionized water to the Eppendorf tube, shaken, and then stored at 30 °C for 30 min. To make an ‘inactivated’ yeast aliquot the suspension was stirred for 2 h in a covered beaker at a temperature of over 80 °C.
A glass slide, covered with semiconducting fluorine-doped tin oxide (FTO) thin film, was used as a substrate for the immobilization of the cells. Before using, FTO/glass has been cleaned following the procedure: (i) sonication in 2% “Micro 90” solution from Sigma Aldrich (Stockholm, Sweden) for 10 min, (ii) rinsing and sonication in deionized water for 10 min, (iii) rinsing with deionized water and sonication in 2-propanol (99.5%, Honeywell) for 10 min, (iv) washing with deionized water and drying in a stream of N2 gas.
For yeast cell immobilization on the FTO surface, poly-L-lysine was used since it improves the attachment of cells to the surfaces. A small drop of poly-L-lysine was placed in the middle of a clean FTO/glass plate and left to dry. Then, 0.5 μm of yeast suspension was placed on the top of the dried poly-L-lysine and left to dry. Afterward, immobilized yeast cells were washed with buffer solution three times to eliminate all the cells that were not immobilized on the surface.

2.3. Electroporation of immobilized cells and electrochemical impedance measurements

Electroporation of immobilized cells was performed by using chronoamperometry while applying 2 V for 120 s to the ⌀ 25 μm Pt UME. To avoid undesired cell damage and electrode contamination with yeast cells, electroporation has been performed while holding UME at a constant 20 μm distance from the surface of immobilized yeast cells.
To capture the electrochemical changes caused by electroporation of yeast cells the electrochemical impedance spectroscopy (EIS) measurements were made: (i) before the electroporation; (ii) 1 min after the electroporation; (iii) 30 min after the electroporation; (iv) 60 min after the electroporation. Registered data were evaluated using analysis software ‘ZView’ by applying a selected equivalent circuit model for the determination of electrochemical system characteristics. The impedance of the system has been registered with the same working electrode and in the same electrode position at which electroporation was performed. EIS spectra were measured in potentiostatic mode, and the range of alternating current frequencies varied from 0.1 Hz to 100 kHz. All the EIS spectra are presented as Nyquist plots, where x-axis represents the Z′, which is real impedance component, and y-axis – Z″, which is imaginary impedance component.

2.4. The assessment of the electroporated area

To determine the electrical impulse affected area size, horizontal scans above the yeast cell surface have been recorded in SECM feedback mode: (i) before the electroporation; (ii) after the electroporation. Both scans have been performed in the same line and direction with a scan rate of 10 μm s-1 and a step of 10 μm at a constant 20 μm distance from the surface of yeast cells.

3. Results and Discussion

⌀ 25 μm Pt ultramicro-electrode has been chosen as a working electrode for the irreversible electroporation process since Pt is biocompatible, inert, and very stable metal (Vižintin et al., 2020), which already showed promising results in irreversible electroporation process for cancer treatment (Lim et al., 2021). Considering that SECM-based and EIS-based investigations of yeast cells can be successfully performed at appropriate potentiostatic conditions (Poderyte et al., 2022; Valiūnienė et al., 2020b), cyclic voltammetry curves were registered to determine a suitable potential value for further SECM and EIS experiments using Pt-based UME as a working electrode.
Cyclic voltammograms of the ⌀ 25 μm Pt ultramicro-electrode in buffer solution, pH 7.1, containing 10 mM of glucose (Fig. 1) show no redox peaks indicating that the electrode is clean, and any unwanted electrochemical reactions occur on the electrode in the potential range from −0.6 V to 0.9 V. Therefore, the potential value for further EIS and SECM measurements has been selected at the Faradaic region of measured current (−0.75 V) because: (i) SECM method is based on measuring the Faradaic current resulting from the electrochemical reaction at the UME; (ii) EIS registration at potentiostatic mode requires controlled potential in the Faradaic current zone as well.

3.1. The evaluation of active yeast cells and the efficiency of electroporation process by EIS-based measurement

To elucidate the possible effect of yeast cells viability on the electrochemical behaviour of the UME, the electrochemical impedance spectra have been recorded placing the ⌀ 25 μm Pt UME at the distance of 20 μm from both active and inactivated yeast cell surface. To keep yeast cells active, 10 mmol of glucose was added to the buffer solution. During all experiments with active yeast cells, glucose was used as an energy source, resulting in yeast cells' metabolism being active and traceable with EIS. The EIS data are presented in a Nyquist plot (Real impedance, Z′ vs Imaginary impedance, Z″) in Fig. 3. The characteristic shape of semicircles appearing in the high-frequency range and representing charge transfer resistance indicates that the charge transfer resistance of the UME above active yeast cells is lower (Fig. 3A, spectrum 1) than the charge transfer resistance above inactivated yeast cells (Fig. 3A, spectrum 2). Thus, it can be concluded that the total impedance above active yeast cells significantly differs from the impedance above inactivated yeast cells. This difference may occur due to the load-bearing substances that are formed during metabolic processes in living (active) yeast cells. Inactivated yeast cells do not undergo metabolic processes, so there are fewer substances that can carry a charge directly to the UME surface (Valiūnienė et al., 2020b). Since it was considered that active yeast cells should generate a higher electrochemical signal after electroporation due to yeast habitual processes that could be interrupted by an electrical pulse, further yeast cell electroporation investigation has been performed only with active yeast cells. Also, electroporation can cause cell destruction or death, thus, during the experiments with initially inactivated yeast cells, significant electrochemical responses are not expected.
Fig. 3
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Fig. 3. A) Electrochemical impedance spectra of the ⌀ 25 μm Pt UME positioned at 20 μm distance from active (1) and inactivated (2) yeast cells, registered in buffer solution, pH 7.1, containing 10 mM of glucose. Applied potential: −0.75 V vs Ag/AgCl,Cl-sat electrode, frequency range 0.1 Hz–100 kHz. B) The equivalent circuit model used for the fitting of EIS data; C) Active yeast cells electroporation performed with ⌀ 25 μm Pt UME, while applying 2 V for 120 s; electrochemical impedance spectra registered placing UME over the yeast cells before the electroporation (curve 1); 1 min after the electroporation (curve 2); 30 min after the electroporation (curve 3); 60 min after the electroporation (curve 4). Measured in buffer solution, pH 7.1, with 10 mM of glucose. UME potential of −0.75 V vs Ag/AgCl,Cl-sat electrode, frequency range 0.1 Hz–100 kHz; D) Magnified data within the range of 0.5 Ω cm2 on the x and y axis representing Z′ and -Z″ data, respectively, that were fitted with the equivalent circuit model.

Contrary to the conventional electroporation model where cell membranes are affected by high voltage (approx. 1 kV cm−1) induced in a two-electrode system (Rems and Miklavčič, 2016), in this research the surface of non-polarized yeast cells was affected by relatively low voltage (2.0 V vs Ag/AgCl,Cl-sat) of the UME in the three-electrode system. The electroporation was performed with SECM by fixing the UME at a 20 μm distance from the active yeast cells' surface and by applying a constant potential of 2.0 V between the UME and the reference Ag/AgCl,Cl-sat electrode for 120 s. The main advantage of selected experimental conditions is the possibility to avoid unwanted damage to active yeast cells that inevitably occurs at high UME voltage.
As can be seen in Fig. 3C, the shapes of obtained spectra of electrochemical impedance are composed of several semicircles. The simplest and the most common Randles equivalent circuit model was selected for the fitting of the data at the high-frequency range displaying the first semicircle (Fig. 3D), because the load transfer resistance is defined from the higher frequency range (Randviir and Banks, 2013). This equivalent circuit is presented in Fig. 3B) and it includes the electrolyte resistance (R0), charge transfer resistance (Rct) that represents the charge-transfer efficiency at the interface between the UME and the electrolyte, and constant phase element (CPE) that represents a capacitance related to the electric double layer (EDL). Since the experimental data (Fig. 3C and D, symbols) coincide well with the fitted data (Fig. 3 D, lines) it was concluded that values of the equivalent circuit elements (Table 1) are fitting appropriately.

Table 1. The values of the equivalent circuit (Fig. 3B) registered by analyzing EIS data of ⌀ 25 μm Pt UME (Fig. 3D).

Empty CellR0,
Ω cm2		CPE mF cm2n (CPE)Rct,
Ω cm2
Before the electroporation0.0029.670.770.15
1 min after the electroporation0.01615.380.840.32
30 min after the electroporation0.01612.220.860.51
60 min after the electroporation0.01611.410.870.50
Results presented in Table 1 illustrate that the CPE of the system resulted in extremely high values reaching 15 mF cm2 1 min after the electroporation. The current between the UME and counter electrode is flowing through two main pathways: directly through the electrolyte solution and through the not-connected investigative surface (Baranski and Diakowski, 2004; Poderyte et al., 2022). As a result, the CPE represents not only electrical double layer capacitance at the UME-PBS interface but also a certain charge storage phenomenon of FTO/glass covered with the yeast cells resulting in high heterogeneity of the surface.
The most important observation of this research was determined by analyzing the values of charge transfer resistance before and after the electroporation procedure. The data determined before the electroporation illustrate (Table 1) that the value of Rct was equal to 0.15 Ω cm2, however, it increases up to 0.32 Ω cm2 1 min after the electroporation and keeps increasing until it finally settles the value of 0.5 Ω cm2 30 min after the electroporation. This observation suggests that irreversible electroporation of the yeast cells has been recorded since the system does not return to its original state, as opposed to the data obtained in previous research where reversible electroporation of the yeast cells was carried out using the Ø 10 μm Au UME (Poderyte et al., 2022). The irreversible increase in the impedance of the Ø 25 μm Pt UME indicates that electrical pulse of 2 V for 120 s resulted in irreversible electroporation of the yeast cells and caused the disruption of the plasma membrane. Contrary to our previous experiments of reversible electroporation when the ions from the yeast cells are released into the solution what is resulting into a decrease of charge transfer resistance, the experiments of this research indicate that proteins and membrane lipids are detached from the plasma membrane and block the surface of the UME (Fig. 1.) resulting in an increase of charge transfer resistance (Table 1). Considering that affected area size while modifying the substrate with SECM depends upon used UME size (Danieli and Mandler, 2013; Grisotto et al., 2011; Matrab et al., 2008; Oswald et al., 2022; Valiūnienė et al., 2020a; Vaske et al., 2021), it is believed that by using the Ø 25 μm Pt UME the pores of critically large size are formed in the plasma membrane. Consequently, if the pores are formed of critically large size, they start to expand instead of resealing until the membrane is disrupted resulting in irreversible electroporation (Fig. 1). Meanwhile, our previous study (Poderyte et al., 2022) showed that if the pores in yeast cells membrane do not reach a critically large size, then they are able to reseal resulting in reversible electroporation.

3.2. The determination of the diameter of electroporated area of yeast cell modified surface

Irreversible electroporation of yeast cells is a very promising technology for biomedical applications, particularly, in cancerous tissue treatment. While performing irreversible electroporation – the cells of the tissue would be damaged/killed without any drugs and/or complicated medical surgery (Agnass et al., 2020; Kwon et al., 2021; Saini et al., 2019; Szlasa et al., 2020). During treatment of cancerous tissues, very often it is crucially important to control the affected area and protect from damages healthy tissues. For this reason, additional experiments to elucidate the diameter of the voltage-affected area have been performed in this research.
To determine the diameter of the UME affected area of yeast cells, the vertical scan above yeast cells was scanned before and after the electroporation procedure performed while applying to the Ø 25 μm Pt UME 2 V voltage for 120 s. The active yeast cells were first scanned before the electroporation (Fig. 4, curve 1). Then the location where the electroporation will be performed was selected from the resulting curve (Fig. 4, dashed line 3). After the electroporation, the UME was returned to the first scan position and the surface scanning was performed again (Fig. 4, curve 2). The surface scans obtained are non-linear due to the yeast being immobilized irregularly on the FTO/glass and their unequal size. Comparing the scan-line registered before (Fig. 4, curve 1) and the scan-line registered after the electroporation (Fig. 4, curve 2) a clear current drop is observed at the electroporated location. Although it is difficult to establish clear boundaries of the area affected by the electrical pulse, however, it is seen the curvature of the curve between ∼200 μm and ∼700 μm (Fig. 4, curve 2), is making the electroporated area with a width approximately of 500 μm. The changes in the surface scan current curve also can be used as proof of the successful electroporation process, since the current recorded above irreversibly electroporated cells was clearly lower than above active yeast cells. Such observations can be correlated to loss of metabolic processes after irreversible electroporation, which is causing yeast cell death.
Fig. 4
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Fig. 4. Surface scan-line of immobilized yeast cells (curve 1) before the electroporation; (curve 2) scan-line after the electroporation. Curve 3 represents location where the electroporation was performed. The scan was performed by using ⌀ 25 μm Pt UME at a constant distance of 20 μm from the surface of yeast cells. UME potential of −0.75 V vs Ag/AgCl,Cl-sat. Measurements were made in buffer, pH 7.1, with 10 mM of glucose. Electrode pitch – 10 μm, horizontal movement speed – 10 μm s−1.

Assuming the obtained results (Fig. 4, curves 1 and 2) the diameter of the affected area has been ∼20 times larger than the diameter of the UME used for the electroporation. Similar results were obtained when a 25 μm diameter Pt-based UME was kept at 8 μm from the gold surface which was patterned with organic compounds, and the affected area resulted in 450 μm diameter (Matrab et al., 2008). Another comparable observation was made while electroporating FTO/glass modified with a hybrid bilayer membrane (Valiūnienė et al., 2020a) when Ø 25 μm Pt electrode was held at 5 μm and the electroporation was performed by applying 1 V for 90 s. This resulted in a formed pore with a diameter of 400 μm. Taking into consideration that a larger diameter of the electroporated area was obtained in this research (500 μm) by using Ø 25 μm Pt UME positioned at a constant distance of 20 μm above the surface, it can be concluded that the diameter of the electroporated area depends on both (i) the diameter of the UME and (ii) the distance between the UME and cell-modified surface (Fig. 5).
Fig. 5
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Fig. 5. The dependence of the diameter of the electroporated area on the distance between the UME and the sample: (i) point 400 μm – Ø 25 μm Pt UME was held at a distance of 5 μm from hybrid bilayer membrane and electroporation was performed by applying 1.0 V potential for 2 min – this resulted in a formed electroporated area with a diameter of 400 μm (Valiūnienė et al., 2020a); (ii) point at 450 μm – Ø 25 μm Pt-based UME was kept at a distance of 8 μm from the gold surface, which was patterned with organic compounds, in this case the affected area resulted in 450 μm diameter (Matrab et al., 2008); (iii) point at 600 μm – Ø 25 μm Pt UME was held at a distance of 20 μm from yeast surface and electroporation, which was performed by applying 2.0 V for 2 min, what resulted in a formation of electroporated area with a diameter of 600 μm (This research).

It can be concluded that the diameter of the electroporated area depends on both (i) the diameter of the UME applied for the electroporation and (ii) the distance between the UME and the surface. Both these findings are in agreement with previously published or (Valiūnienė et al., 2020a) and data reported by some other researchers (Matrab et al., 2008).

4. Conclusions

In this research irreversible electroporation of the yeast cells has been achieved by applying a 2.0 V (vs Ag/AgCl,Cl-sat) electrical pulse to the Ø 25 μm Pt UME positioned at a 20 μm distance from the surface of active yeast cells. The results of electrochemical impedance spectroscopy showed that the charge transfer resistance at the interface between the Ø 25 μm Pt UME and the electrolyte increases irreversibly after the electroporation procedure, which indicates that the surface of the UME was blocked by proteins and membrane lipids detached from the plasma membrane during the electroporation. An electrical pulse of 2.0 V lasting for 120 s causes the disruption of the plasma membrane and results in irreversible electroporation of the yeast cells making the electroporated area with a diameter of approximately 500 μm. The diameter of the electroporated area is ∼20 times larger than the diameter of the UME used for the electroporation. It can be concluded that the diameter of the electroporated area depends on both (i) the diameter of the UME applied for the electroporation and (ii) the distance between the UME and the surface.

CRediT authorship contribution statement

Margarita Poderyte: Methodology, Investigation, Formal analysis, Writing – original draft, Interpretation of data, Formal analysis. Arunas Ramanavicius: Interpretation of data, Formal analysis, Supervision, Conceptualization, Writing – review & editing, Funding acquisition. Aušra Valiūnienė: Methodology, Investigation, Formal analysis, Writing – original draft, Interpretation of data, Formal analysis.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Ramanavicius reports financial support was provided by Research Council of Lithuania.

Acknowledgement

This project has received funding from the Research Council of Lithuania (LMTLT), agreement No [S-MIP-20-18].

Data availability

Data will be made available on request.

References

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