Journal of Electroanalytical Chemistry 878 (2020) 114572

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Journal of Electroanalytical Chemistry

journal homepage: www.elsevier.com/locate/jelechem

Electrochemical impedance spectroscopy study of liposome adsorption and

rupture on self-assembled monolayer: Effect of surface charge
Yusuke Fujino a, Ryo Nakamura a, Huan-Wen Han b, Ichiro Yamashita b, Tomohiro Shimizu a,
⁎
Shoso Shingubara a, Takeshi Ito a,
a
Graduate School of Science and Engineering, Kansai University, Yamatecho 3-3-35, Suita, Osaka 564-8680, Japan
b
Graduate School of Engineering, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan

A R T I C L E I N F O A B S T R A C T

Article history: We report here that the selection of the electrode surface charge is very crucial in evaluating the adsorption of lipo-
Received 24 June 2020 somes and their ruptures by means of electrochemical impedance spectroscopy (EIS). We prepared three types of elec-
Received in revised form 7 August 2020 trodes whose surface characteristics were controlled by self-assembled monolayers (SAMs). The chemicals for SAM
Accepted 8 August 2020
contained the same straight-chain alkanes with thiol groups at one end to bind with the Au electrode surface. The
Available online 11 August 2020
head groups were CH3 for 1-dodecanethiol (DDT), CH2OH for 11-mercapto-1-undecanol (MUD), and CH2NH2 for
11-amino-1-undecanthiol (11-AUT). The surface charges were neutral for DDT, negative for MUD, and positive for
11-AUT. Resulting from liposome adsorption and rupture, the Nyquist plots changed differently, depending on the sur-
face charges. We concluded that the easiest and most sensitive surface to handle was obtained by 11-AUT SAM. The EIS
measurements, obtained from the electrode containing 11-AUT SAM, successfully estimated the critical micelle con-
centration (CMC) of the surfactant, Triton X-100 through the observation of the liposome rupture process.

1. Introduction Another important liposome research field is DDS where liposomes

with diameters, ranging from several tens of nanometers to several micro-
Because liposomes could mimic the lipid bilayer membranes of cells, meters, were designed to encapsulate desired amounts of drugs, attain tar-
they have attracted increased attention from researchers since the last get tissues or locations, and release the drugs at the timely manner. There
50 years [1–4]. Liposome possesses a spherical-shaped shell that is con- are three important aspects of liposome-based DDS. Firstly, the drug must
structed from amphiphilic molecules. Its outer and inner layers are com- be successfully encapsulated within the liposome. Secondly, it must be spe-
posed of hydrophilic and hydrophobic molecules. Further, the size of cifically adsorbed to the target. At last, it must be ruptured to release the en-
liposome varies from several tens of nanometers to sub-millimeters. The re- capsulated drug to the target. All the processes must be precisely controlled.
search field of liposomes is wide, ranging from artificial cells to drug deliv- The combination of artificial cell mimic and DDS technology could afford
ery systems (DDSs). Some researchers have employed liposomes for the much information through experimental drug trials with high throughputs.
elucidation of the life activities of a cell and prepared quasi-cells, which Hence, the handling of liposomes is essential. Drug encapsulation is rela-
could move autonomously, change their shapes, and undergo cell division tively easy to achieve by introducing the drug into the solution during lipo-
[5–8]. Additionally, liposomes are promising materials in clinical screen- some formation. The hard issues are controlling the adsorption of the
ing. Although a developed drug requires animal tests to determine its effi- liposome onto the target selectively and the release of the drug by its own
cacy in disease improvement before clinical trials, it is ethically preferable rupture [9–13]. The raised issues are actually quite diverse, but our study
to avoid them [14,15]. Recently, some researchers started utilizing artificial is surely one of trials to clarify the basis for it. Namely, the electrostatic in-
cells (deoxyribonucleic acids (DNAs) were inserted in the liposome, and teraction is key factor to watch liposome adsorption and liposome destruc-
some molecules were added to the liposome surface to bind with other ma- tion by using EIS.
terials) instead of animals. [16–18].

Abbreviations: DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DDT, 1-dodecanethiol; 11–AUT, 11-amino-1-undecanthiol; 3-MPA, 3-mercaptopropionic acid; Rct, charge transfer resistance;
CE, counter electrode; CV, cyclic voltammetry; DNA, deoxyribonucleic acid; Z, complex impedance; CMC, critical micelle concentration; DDS, drug delivery system; EIS, electrochemical impedance
spectroscopy; Z′, impedance; ITO, indium tin oxide; LSPR, localized surface plasmon resonance; MUD, mercapto-1-undecanol; PBS, Phosphate buffered saline; SAM, self-assembled monolayer;
Triton X-100, polyethylene glycol mono-p-isooctylphenyl ether; Rs, solution resistance; SERS, surface-enhanced Raman spectroscopy; SPR, surface plasmon resonance; QCM-D, quartz crystal mi-
crobalance with dissipation; RF, radio frequency; RE, reference electrode; WCA, water contact angle; WE, working electrode.
⁎ Corresponding author.
E-mail address: [email protected]. (T. Ito).

http://dx.doi.org/10.1016/j.jelechem.2020.114572
1572-6657/© 2020 Elsevier B.V. All rights reserved.
Y. Fujino et al. Journal of Electroanalytical Chemistry 878 (2020) 114572

To achieve such aims, we first comprehensively observed the adsorp- surface. The most sensitive surface was obtained when 11–AUT was uti-
tion and rupture of liposomes. However, a spherical liposome with a diam- lized. EIS measurements employing an electrode with 11-AUT SAM suc-
eter of 1 μm possesses only 0.52 fL inner space, indicating that highly cessfully estimated the critical micelle concentration (CMC) of the
sensitive measurement methods were required. Thus far, many studies surfactant, Triton X-100.
have been conducted, employing quartz crystal microbalance with dissipa-
tion (QCM-D) methods [19–22] and plasmonic techniques, such as the sur-
face plasmon resonance (SPR), localized surface plasmon resonance (LSPR) 2. Materials and methods
and surface-enhanced Raman spectroscopy (SERS) [20–24], and electro-
chemical impedance spectroscopy (EIS) [19,22,25–28] measurements. 2.1. Materials
Among them, EIS is easy and highly sensitive to the surface condition of
an electrode. Here, we, therefore, focused on EIS as the observation tool Phosphate buffered saline (PBS, pH = 7.4), sodium chloride (NaCl),
for the adsorption and rupture of liposomes. polyethylene glycol mono-p-isooctylphenyl ether (Triton X-100), potassium
EIS detects changes in the capacitance and resistance of an electrode hexacyanoferrate (II) (potassium ferrocyanide, K4[Fe(CN6)]), and potas-
surface due to biochemical reactions. When biorecognition events, such sium hexacyanoferrate (III) (potassium ferricyanide, K3[Fe(CN)6]) were
as antigen–antibody interactions and/or physical adsorptions, occur, EIS purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka,
could detect them as soon as the surface impedance (Z′) changes. The com- Japan). Further, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (NOF
plex impedance (Z), measured by EIS, is presented as the sum of the real Corp., Tokyo, Japan), a well-known chemical in liposome preparation,
(Zreal) and imaginary impedances (Zimg). The plots of -Zimg vs. Zreal, at a was employed in this study [30,31]. A syringe filter, with a 220 nm pore
given frequency, are called the Nyquist plots, which display a semicircle. membrane, and a 2.5 mL syringe were purchased from AS ONE, Inc.,
The solution resistance (Rs) of the measurement system is presented at Japan. The selected chemicals for the fabrication of SAM were DDT,
the left end of the semicircle. The diameter of the semicircle represents MUD, and 11-AUT with HCl, which were obtained from Sigma Aldrich
the charge transfer resistance (Rct) on the electrode surface. Rct represents Co., LLC. Sputtering targets of Au (99.999%) and indium tin oxide (ITO)
the reactivity of the electrons around the electrode surface. In the simplest were purchased from Tanaka Kikinzoku Kogyo K. K. (Tokyo, Japan) and
electrochemical reaction model, Z is the sum of Rs and Z′ due to a parallel Kojundo Chemical Laboratory Co., Ltd. (Saitama, Japan), respectively.
circuit of double-layer capacitance (Cdl) and Rct, according to the following
equations [29]:
2.2. Electrochemical measurements
Z ¼ Rs þ Z0 ð1Þ
A working electrode (WE) was obtained for the electrochemical mea-
0 Rct surements by a conventional radio frequency (RF) sputtering method.
Z ¼ ; ð2Þ
1 þ iωRct C dl After the thermal oxidation of the Si substrate, ITO (20 nm thick) and
Au (100 nm thick) were deposited on the SiO2/Si substrate in order.
where i is an imaginary number. In this study, Z' was expected to change ac- Here, the ITO layer was used as an adhesive layer, because it has good
cording to the adhesion and rupture (removal) of liposomes. adhesion, good film formation, low electrical resistivity, and smooth sur-
To the best of our knowledge, only a few reports exist on the effect of face without external heating on the deposition [32]. We utilized a con-
surface charge on liposome adsorption and rupture. Yan et al. [25] reported ventional three-electrode system composed of an Ag/AgCl (3 M NaCl)
that a negatively charged surface, coated with a self-assembled monolayer reference electrode (RE), a Pt wire counter electrode (CE), and an Au
(SAM) of 3-mercaptopropionic acid (3-MPA), depressed the redox reaction thin film-coated WE. RE, CE, and the measurement cell (1 mL) were pur-
of [Fe(CN)6]3−/4− and that divalent cations, such as Mg2+, Ba2+, and chased from BAS Inc., Tokyo, Japan. The diameter of WE was set at
Ca2+, coated with SAM, improved the redox reaction. This finding affords 6 mm, which was defined by the O-ring, located at the bottom of the
an insight into improving the sensitivities of the adhesion and rupture of measurement cell. Potassium ferrocyanide (1 mM) and potassium ferricy-
liposomes. anide (1 mM) were employed as redox probes and were dissolved in PBS
Here, we report the effect of the surface charge, produced by SAM, on containing NaCl (150 mM), as the electrolyte, to obtain an electrolyte so-
electrochemical reactions. Specifically, we utilized 1-dodecanethiol lution. Cyclic voltammetry (CV) and EIS were performed with electro-
(DDT), 11-mercapto-1-undecanol (MUD) or 11–hydroxy–1–undecanthiol chemical measurement equipment (HZ–7000, Hokuto Denko Co.,
(11–HUT), and 11-amino-1-undecanthiol (11-AUT) for as SAM. These Tokyo, Japan). The potential range on the CV instrument was 0–0.5 V
chemicals possess the same straight-chain alkanes with thiol groups to vs. RE with a sweep rate of 50 mV/s. The frequency range of EIS was
bind with the Au surface. Moreover, the head groups were different: DDT 100 KHz–0.1 Hz with an AC amplitude of 10 mV rms to the spontaneous
possesses CH3, which affords it a neutrally charged hydrophobic surface; potential.
MUD possesses CH2OH, which affords it a hydrophilic surface that is nega- The measurement protocol is described as follows:
tively charged; and CH2NH2 in 11–AUT affords a surface that is hydrophilic
and positively charged. 1. After 1 mL of the electrolyte solution was injected into the cell, the CV
We observed that the head group of SAMs affected the detection of lipo- and EIS experiments were performed to evaluate the initial state of the
some adsorption and rupture. It was also clear that the surface charge is electrode.
very important in the evaluation of liposome behavior on the electrode 2. After washing the cell with distilled water, a solution containing lipo-
some (volume: 20 μL), was dispensed into the cell and kept for
30 min. At this time, liposome adhered to the electrode surface.
Table 1 3. Further, after washing the cell with distilled water, 1 mL of the electro-
Characteristics of the four types of electrodes employed in this study. lyte solution was dispensed into the cell. Thereafter, CV and EIS were
Electrode types Head group of SAM Charge of the head group WCA⁎ (°) performed to evaluate the electrode condition after liposome adhesion.
4. After washing the cells with distilled water, 1 mL of a detergent-
Type A – – 84.1
Type B CH3 Neutral 105 containing solution (1 wt% Triton X-100) was dispensed into the cell
Type C CH2OH Negative 14.7 and kept for 3 min to break the liposomes.
Type D CH2NH2 Positive 68.4 5. After washing the cell with distilled water, 1 mL of the electrolyte solu-
WCA⁎ is the water contact angle. WCAs shown here are the average of five measurements tion was dispensed into the cell. CV and EIS were performed to evaluate
on each sample. Photos on the WCA evaluation are displayed in Fig. 1S. the electrode condition after the rupture of the liposome.

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Y. Fujino et al. Journal of Electroanalytical Chemistry 878 (2020) 114572

Fig. 1. (a) Cyclic voltammograms and (b) Nyquist plots of bare Au (red), after liposome adhesion (blue), and after Triton X-100 (green) addition. The electrode diameter was
6 mm and the sweep rate was 50 mV/s of CV. The EIS measurements were performed at a frequency of 100 KHz–0.1 Hz.

2.3. Surface modification of WE 2.4. Formation of liposome

We prepared four types of WE. The surface wettability of these sur- Firstly, 1.5 mg of DOPC was dissolved in ethanol (1 mL), after which
faces was varied as shown in Table 1. Type A was coated with Au/ITO the solvent was dispensed in a microtube (1.5 mL). The microtube was
thin films on the SiO2/Si substrate, as a reference. Type B was coated placed in a thermal chamber for 24 h to vaporize ethanol during which
with SAM of DDT on the Au thin film and exhibited hydrophobicity. the temperature of the chamber was kept at 50 °C. Afterward, a thin or-
Types C and D were coated with SAMs of MUD and 11–AUT on the Au ganic film was formed along the inner surface of the microtube. After dis-
thin films, respectively. These electrodes exhibited hydrophilicity but tilled water was added to the microtube, it was sonicated for 5 min to
their surface charges were different; Type C was negatively charged and form the liposome, by peeling the organic film off, following the vortex
Type D was positively charged. All the electrodes with SAM were pre- of the microtube for 1 min. Finally, we obtained a solution containing
pared by dipping Type A into the ethanol solution, dissolved with each DOPC with a diameter of ˂ 220 nm because the solution was filtered
SAM chemical for 24 h. 20 times through the syringe filter.

Fig. 2. Graphs of (a) cyclic voltammograms and (b) Nyquist plots of bare Au (red) and methylated the SAM-coated electrode (blue). The inset graph is an enlarged view of the
voltammograms of the methylated SAM-coated electrode. Graphs of (c) CVs and (d) Nyquist plots of the methylated SAM-coated electrode after liposome adhesion (blue) and
after the addition of Triton X-100 (green). The electrode diameter was 6 mm, the sweep rate was 50 mV/s of CV. EIS measurements were performed at a frequency of 100
KHz–0.1 Hz.

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Y. Fujino et al. Journal of Electroanalytical Chemistry 878 (2020) 114572

Fig. 3. Graphs of (a) cyclic voltammograms and (b) Nyquist plots of bare Au (red) and hydroxylated SAM-coated (blue) electrodes. Graphs of (c) CVs and (d) Nyquist plots of
the hydroxylated SAM-coated electrode after liposome adhesion (blue) and after the addition of Triton X-100 (green). The electrode diameter was 6 mm, the sweep rate was
50 mV/s of CV. The EIS measurements were performed at a frequency of 100 KHz–0.1 Hz. (e) Illustration of the interaction and surface charge, based on liposome adsorption
and rupture of Type C.

3. Results and discussion ~105°. Fig. 2(a) and (b) show the cyclic voltammograms and Nyquist
plots before (red lines) and after (blue lines) the SAM coating. Rct in-
3.1. Au surface creased drastically and the current in the voltammograms reduced drasti-
cally after the coating. The calculated Rct values before and after the
The cyclic voltammograms and Nyquist plots of the Au electrode (Type coating were 128 Ω and 16.8 MΩ, respectively. The methylated surface
A) are shown in Fig. 1. Because the Au surface was clean and uninterrupted exhibited hydrophobicity and was neutrally charged, thereby resulting
by the adsorbed material, the semicircle in the Nyquist plot was the in a low affinity between the electrode and electrolyte solution since
smallest, and the oxidation current on the voltammograms was the largest. the redox probes ([Fe(CN6)]3−/4−) were negatively charged. These char-
The potential between the oxidation and reduction current peaks (ΔEp) was acteristics complicated the transfer of electrons and resulted in a signifi-
~70 mV. After the liposomes had adhered to the electrode, the semicircle in cant Rct increase.
the Nyquist plot increased because Rct had been increased by the steric bar- Fig. 2(c) and (d) show the voltammograms and Nyquist plots before
rier of the liposome, which interrupted electrochemical charge transfer. As and after the adsorption and rupture of the liposomes on Type B. After
Rct increased, the oxidation current peak decreased and to the least. After the adsorption of the liposomes, Rct slightly increased and the current
breaking the liposomes, the semicircle on the Nyquist plot decreased, al- in the voltammogram decreased. The amount of liposome, which adhered
though it did not return to the initial state. Similarly, the peak of the oxida- to the CH3 surface was small because the electrode surface exhibited hy-
tion current increased, compared to the case of liposome adhesion. drophobicity while the liposome exhibited hydrophilicity. The changes in
However, it was smaller than that of the initial state. Additionally, ΔEp in- the electrochemical signals might be due to the adsorption of fragments
creased slightly. These data show that the ruptured liposomes and/or sur- of the liposomes, which were not spherical but displayed a planar surface.
factants had adhered to the electrode surface. Such slight liposome adsorption might work as an insulator to inhibit
electron transfer. After adding the surfactant, Rct decreased and the cur-
3.2. Hydrophobic surface rent in the voltammogram increased. Here, the surfactant possessed
both hydrophobic and hydrophilic groups. Thus, the surfactant could ad-
When the electrode surface was modified with DDT (Type B), hydro- here to the methylated surface, which utilized its hydrophilic group, via
phobicity due to the CH3 group was obtained. WCA of the surface was the hydrophobic group.

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Y. Fujino et al. Journal of Electroanalytical Chemistry 878 (2020) 114572

Fig. 4. Graphs of (a) CVs and (b) Nyquist plots of bare Au (red) and –NH2 group SAM-coated electrode (blue). Graphs of (c) CVs and (d) Nyquist plots of the –NH2 group of the
SAM-coated electrode after liposome adhesion (blue), and the addition of Triton X-100 (green). The electrode diameter was 6 mm, the sweep rate was 50 mV/s in CV. The EIS
measurements were performed at a frequency of 100 KHz–0.1 Hz. The (e) Interaction and surface charge of liposome adsorption and rupture on Type D.

3.3. Hydrophilic surface increased slightly and the reduction current also increased in CV. The mag-
nitude of the reduction current was somewhat larger than that of the oxida-
We also prepared two hydrophilic surfaces, Types C and D. Type C was tion current. These characteristics were caused by the surface charge
coated with MUD. The surface contained hydroxyl (–OH) groups and was alternation after liposome adsorption, as shown in Fig. 3(e). As mentioned
negatively charged. Further, Type D was coated with 11–AUT. The surface above, the –OH surface was negatively charged, thus repelling the redox
contained amino (−NH2) groups and was positively charged. probes. After liposome adsorption, the surface charge became positive be-
Firstly, we discussed the results for Type C. Next, Fig. 3(a) and (b) show cause the surface of DOPC was positively charged. Thus, the redox probes
the voltammograms and Nyquist plots before (red lines) and after (blue were attracted by an electrostatic attractive interaction, and Rct decreased.
lines) the SAM coating. It is clear that Rct increased substantially and that After adding the surfactant, Rct increased, almost back to that, before lipo-
the current of the voltammogram was reduced drastically after the coating. some adsorption. The voltammograms before liposome adsorption and
The –OH surface exhibited hydrophilicity and was negatively charged, thus after the addition of the surfactant exhibited almost the same behavior.
resulting in a low affinity between the electrode and the electrolyte solu- These characteristics are completely different from those of Type A, a nor-
tion. The –OH surface repelled [Fe(CN6)]3−/4− due to like-charge polarity. mal Au electrode.
This property was almost reported by Wang et al. which introduced a Next, we discussed the results of Type D, which was a positively
graphene-modified glassy carbon electrode (GCE) to detect target DNA charged surface. Fig. 4(a) and (b) show the voltammograms and
[33]. HP-probes, SS-probes and dNTPs in the reaction solution adsorbed Nyquist plots before (red lines) and after (blue lines) SAM coating.
on the GCE through the strong π-π stacking between nucleotides and The voltammograms of Type D are slightly different from those of
GCE, which made Rct increase due to the electrostatic interaction between Types A–C. There were oxidation and reduction current peaks even
redox probes (Fe(CN)3− 4−
6 /Fe(CN)6 ) and nucleotides since they were nega- though ΔE p (100 mV) was broad. Additionally, the increase in R ct
tively charged. Although there was an electrostatic repulsive force, Rct of of Type D was minimal after the SAM coating, compared to that of
Type C was much smaller than that of Type B because SAM exhibited those in the other electrode types. These results indicate that the –
hydrophilicity. NH2 surface exhibited a good affinity for the [Fe(CN6)]3−/4– redox
Fig. 3(c) and (d) show the voltammograms and Nyquist plots of the ad- probe. The –NH2 surface exhibited hydrophilicity and was positively
sorption and rupture of the liposomes. After the adsorption of the lipo- charged, thereby attracting [Fe(CN6 )] 3−/4− by an electrostatic at-
somes, Rct decreased substantially. Further, the oxidation current tractive force and inhibiting the increase in Rct.

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Y. Fujino et al. Journal of Electroanalytical Chemistry 878 (2020) 114572

decrease in Rct. Rct, after the addition of Triton X-100, decreased inversely
to the Triton X-100 concentration, until around ~0.10 wt%. Since different
WEs with Au thin film were employed in each measurement, and Rct after
SAM coating ranged from 4 to 10 KΩ. For a more precise analysis, we intro-
duced the ratio, |ΔRct(1)|/|ΔRct(2)| as the evaluation parameter. Here, |ΔRct
(1)| and |ΔRct(2)| were obtained as the equations below:

j ΔRct ð1Þ j¼j Rct ðafter liposome adsorptionÞ−Rct ðafter the SAM coatingÞ j

j ΔRct ð2Þ j¼j Rct ðafter liposome adsorptionÞ−Rct ðafter adding Triton X−100Þ j

The parameter, |ΔRct(1)|/|ΔRct(2)|, will be 1.0 when Rct (after the addi-
tion of Triton X-100) returns to the initial Rct, after SAM coating, and it will
be 0.0 when there is no rupture.
Fig. 5 shows |ΔRct(1)|/|ΔRct(2)| versus the concentration of Triton X-
100. |ΔRct(1)|/|ΔRct(2)| increased with the concentration of Triton X-100,
Fig. 5. Variability ratio of Rct, |ΔRct(1)|/|ΔRct(2)| was plotted against the and the value saturated at over 0.10 wt%. The saturated value of |ΔRct
concentration of Triton X-100.
(1)|/|ΔRct(2)| was ~0.9, which implies that the electrode surface almost re-
covered to its initial state. This could be attributed to the adsorption of Tri-
Fig. 4(c) and (d) show the voltammograms and Nyquist plots of the ad- ton X-100, which was indicated by the QCM-D measurement results
sorption and rupture of the liposomes. After the adsorption of the lipo- (Fig. S3). The increment of |ΔRct(1)|/|ΔRct(2)| over the concentration of
somes, Rct increased considerably because of the steric barrier of the 0.01 wt% was ˂˂ the increment below the concentration of 0.01 wt%.
liposomes. In this case, the liposome adhered to the surface via a hydro- From the results, CMC was estimated to range from 0.01% to 0.1%,
philic interaction. The surface charge was unchanged because of liposome which was consistent with the previously reported value 2.4 × 10−4 M
adsorption. Thus, we could directly monitor the interference of electron (0.013 wt%) [34]. Further, we would increase the precision to estimate
transfer on the electrode surface, which was caused by liposome adsorp- CMC by repeating the experiments and isolating the concentration depen-
tion. The voltammograms indicated that the oxidation and reduction dence of the absorption of Triton X-100.
peaks drastically decreased after liposome adsorption. This finding is con-
sistent with the large increase in Rct. After adding the surfactant, Rct de- 4. Conclusions
creased to approximately two times the size of the SAM-coated surface.
Moreover, the magnitudes of the oxidation and reduction current peaks in- We discussed the cyclic voltammograms, EIS, and Nyquist plot charac-
creased. ΔEp, after the addition of the surfactant, was between those before teristics, derived from liposome adsorption and rupture on Au electrodes
and after liposome adhesion. These data demonstrate that the ruptured li- with/without SAM. The Nyquist plots changed according to the adsorption
posomes and/or surfactants had adhered to the electrode surface. and rupture of the liposomes in all the cases. However, the characteristics of
As already discussed, the –NH2 surface was superior in detecting the ad- the changes were different. It is, therefore, clear that multiple factors, such
hesion and rupture of liposomes than the other electrodes, coated with/ as hydrophilicity and surface charge, on the electrode, are very important
without methylated and hydroxylated SAM, when [Fe(CN6)]3−/4− was uti- in improving the sensitivity of EIS. Based on the detailed studies of all the
lized as a redox probe. This superiority results from multiple factors, such as electrodes, it was concluded that the Au electrode with an –NH2 surface
hydrophilicity and positive charge of SAM, which attracted [Fe(CN6)]3−/4 maximized the magnitude of Rct change, corresponding to adhesion and
−
around the electrode surface, as shown in Fig. 4(e). The important notice rupture of the liposomes. The positively charged Au electrode with an –
is that we can chose a suitable electrode surface by changing SAM to detect NH2 surface attracted a redox probe ([Fe(CN6)]3−/4−), which increased
what we want. These results will be useful for basic research to control the the ease of detecting liposome adsorption and rupture. EIS measurements,
liposome adsorption on targets and drug release. The findings in this paper utilizing the Au electrode with an –NH2 surface successfully estimated
will also be useful when building DDS evaluation systems with integrated CMC of the surfactant, Triton X-100, through liposome rupture process
electrode arrays and microfluidic devices. observation.

3.4. Estimation of CMC of Triton X-100 Funding

As discussed in section 3.3, we noticed that Type D was the most sensi- This work was supported by JSPS KAKENHI [Grant Number JP
tive electrode for liposome adsorption and rupture. Thus, we applied Type 18K19008].
D in the measurement of CMC of Triton X-100. It is known that the steric
structure of surfactants, such as Triton X-100, depends on its concentration. Author statement
Triton X-100 is composed of hydrophilic and hydrophobic groups, as
shown in the inset of Fig. 3(e) and in Fig. 4(e). At low concentrations, the Contributors Y. F and R. N were responsible for analyzing all data ob-
surfactant existed as a monomolecular. At CMC, the monomolecular mole- tained from experiments. T. I was the chief investigator and also responsible
cule binds together to form micelles. We determined CMC of Triton X-100 for the data consideration. H\\W. H and I. Y were responsible for the data
by observing the liposome rupture. consideration. T. I, T. S, and S. S were responsible for supervision and pro-
The experimental procedure was the same as that, described in section ject administration. All authors contributed to the writing of the final man-
3.3, except for the varying of the Triton X-100 concentration from 0.0001 to uscript and approved the manuscript to be published. All members of study
0.5 wt%. The Nyquist plots after SAM coating (red line), liposome adsorp- team contributed to the management or administration of this study.
tion (blue line), and addition of Triton X-100 (green line) are presented in
the supplemental information (Fig. S2(a)–(g)). At the lowest concentration Declaration of Competing Interest
(0.0001 wt%), the Nyquist plot before and after the addition of Triton X-
100 exhibited no difference, indicating that liposomes were not ruptured. The authors declare that they have no known competing financial inter-
The addition of Triton X-100 at such a low concentration did not exert ests or personal relationships that could have appeared to influence the
any influence. However, the 0.001 wt% Triton X-100 caused a slight work reported in this paper.

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Y. Fujino et al. Journal of Electroanalytical Chemistry 878 (2020) 114572

Appendix A. Supplementary data [17] Y. Elani, T. Trantidou, D. Wylie, L. Dekker, K. Polizzi, R.V. Law, O. Ces, Constructing
vesicle-based artificial cells with embedded living cells as organelle-like modules, Sci.
Rep. 8 (2018) 4565.
Supplementary data to this article can be found online at https://doi. [18] Y. Yamada, T. Ishikawa, H. Harashima, Validation of the use of an artificial mitochon-
org/10.1016/j.jelechem.2020.114572. drial reporter DNA vector containing a Cytomegalovirus promoter for mitochondrial
transgene expression, Biomater. 136 (2017) 56–66.
[19] E. Briand, M. Zach, S. Svedhem, B. Kasamo, S. Petronis, Combined QCM-D and EIS
study of supported lipid bilayer formation and interaction with pore-forming peptide,
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