Sensors and Actuators B 238 (2017) 1277–1282

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Sensors and Actuators B: Chemical

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

The application of bismuth film electrode for measuring Zn(II) under

less acidic conditions in the presence of cell culture medium and ZnO
nanoparticles
Tea Romih a,1 , Samo B. Hočevar b,∗ , Veno Kononenko a , Damjana Drobne a
a
Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, SI-1000, Ljubljana, Slovenia
b
Department of Analytical Chemistry, National Institute of Chemistry, Hajdrihova 19, SI-1000, Ljubljana, Slovenia

a r t i c l e i n f o a b s t r a c t

Article history: A study for the application of bismuth film electrode (BiFE) under less acidic conditions for anodic strip-
Received 7 July 2016 ping voltammetric measurements of Zn(II) in complex organic sample, i.e. in the cell culture medium
Received in revised form containing ZnO nanoparticles, is presented. The BiFE was prepared in-situ on a substrate glassy car-
15 September 2016
bon electrode in solution containing 0.1 mol L−1 piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES) and
Accepted 16 September 2016
0.1 mol L−1 KNO3 as supporting electrolyte, adjusted to pH 6.5, and in the presence of dissolved oxygen.
Available online 20 September 2016
In the model solution, i.e. in the absence of cell culture medium and ZnO nanoparticles, the BiFE revealed
good linear response in the examined concentration range of 10–100 ␮g L−1 Zn(II) with r2 of 0.994, the
Keywords:
Bismuth film electrode
LOD of 0.14 ␮g L−1 associated with 120 s accumulation step, and favorable repeatability of 1.7%. Upon the
Anodic stripping voltammetry addition of cell culture medium, the signal of Zn(II) attenuated for ca. 64%; however, the BiFE still exhib-
Zinc ited excellent linear response in the examined concentration range of 10–100 ␮g L−1 Zn(II) with r2 = 0.999,
Nanoparticles favorably low LOD of 0.15 ␮g L−1 after 120 s accumulation, and satisfactory repeatability of 3.0%. Finally,
Cell culture medium the applicability of the proposed method was successfully demonstrated through measuring Zn(II) in
the cell culture medium containing 5 mg L−1 of ZnO nanoparticles for the purpose of a nanotoxicological
study.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction of real samples which often feature complex matrices of organic

The research of nanoparticle (NP) solubility and dissolution as a
part of their characterization has attracted great attention in the
recent years [1]. For this purpose, the (stripping) voltammetric
techniques have been recognized as preferential over spectroscopic
ones, since the former obviate the need for relatively tedious and
time-consuming separation of NPs from dissolved metal species.
The stripping voltammetric techniques are also considered as
advantageous over their potentiometric analogues (associated with
ion-selective electrodes), particularly due to lower detection lim-
its of the former [1]. The adopted electrochemical method should
include the operation under conditions that do not affect consider-
ably the original sample, particularly with respect to pH, metal ion
complexation, etc. Such a virtue is non-trivial, especially in the case
∗ Corresponding author.
E-mail address: samo.hocevar@ki.si (S.B. Hočevar).
1
Current address: SEYENS Information Solutions and Education Ltd., Krimska
ulica 20, SI-1000 Ljubljana, Slovenia.
molecules and inorganic solutes.
As a favorable alternative to its mercury counterpart, the bis-
muth film electrode (BiFE) has been introduced in 2000 [2]. In most
studies, the in-situ prepared BiFE has been shown to operate with
its highest electroanalytical performance at pH 4–5, preferentially
in the acetate buffer solutions [3]. However, a few studies corrob-
orated its favorable applicability also in the media with lower and
higher pH values, e.g. upon the addition of mildly chelating agents
such as tartrate [4,5]. The pH of 4–5 might not be optimal for mea-
suring dissolved metal species in real samples containing certain
NPs, such as ZnO NPs, which are one of the most studied NPs in the
nanotoxicological field, but at the same time one of the most soluble
[6]. Dissolved Zn(II) is one of the main factors that govern the tox-
icity of ZnO NPs to organisms, while organism-dependent cellular
uptake of NPs and induction of oxidative stress and consequent cel-
lular damage may also contribute to ZnO NP toxicity [6]. However,
it is necessary to conduct stripping voltammetric measurements of
the dissolved metal species near the pH of real samples, which is
between 6.5 and 7.4 in the majority of biologically relevant cases.

http://dx.doi.org/10.1016/j.snb.2016.09.090
0925-4005/© 2016 Elsevier B.V. All rights reserved.
1278 T. Romih et al. / Sensors and Actuators B 238 (2017) 1277–1282
There exists a variety of organic buffers with the effective buffer-
ing capacity in the circumneutral pH range, collectively known
as “Good’s buffers” after the researchers that introduced them in
1966 [7]; however, through subsequent research it has been discov-
ered that some of these buffers bind metal ions and are therefore
unsuitable for analytical purposes [8]. In the group of the most
auspicious buffers are N-substituted aminosulfonic acids with a
morpholinic or piperazinic ring, for which metal complexation is
negligible or at least limited to specific metal ions [8]. Among these,
piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES) has been suc-
cessfully implemented in the direct measurement of dissolved zinc
in the presence of ZnO NPs by anodic stripping voltammetry using
mercury electrode [9]. The influence of PIPES on redox processes
of zinc has been investigated by differential pulse anodic stripping
voltammetry at the mercury electrode, confirming that it neither
complexes zinc nor modifies the reversibility of its electrochemical
response [10].
In this work, we present a novel method for anodic stripping
voltammetric determination of Zn(II) using the in-situ prepared
bismuth film electrode (BiFE) operating in PIPES buffer solution at
circumneutral pH in the presence of dissolved oxygen and a com-
plex organic matrix containing ZnO NPs. Several key parameters
were optimized to enable rapid and reliable operation for the pur-
pose of measuring Zn(II) in the real sample of commercial ZnO NPs
incubated in the cell culture medium.
2. Experimental
2.1. Reagents and solutions
All chemicals were of the analytical grade purity. The stan-
dard solutions of Bi(III) and Zn(II) were provided by Merck
(Darmstadt, Germany) and further diluted as required. For
anodic stripping voltammetric (ASV) experiments, a mixture
of 0.1 mol L−1 piperazine-N,N -bis(2-ethanesulfonic acid) (PIPES;
Avocado Research Chemicals Ltd, Lancaster, UK) and 0.1 mol L−1
KNO3 (Merck) was used as the supporting electrolyte. The pH was
adjusted to 6.5 with 0.5 M KOH (Kemika Zagreb, Croatia). Water
used throughout the work was first deionized and then further
purified using Elix 10/Milli-Q Gradient unit (Millipore, Bedford,
Massachusetts, USA).
2.2. Cell culture medium preparation
The experimental cell culture medium was prepared as follows:
the mixture of two common cell culture media, i.e. Dulbecco’s mod-
ified Eagle’s medium (Sigma-Aldrich, Steinheim, Germany; product
no. D5671) and Nutrient mixture F-12 Ham (Sigma-Aldrich, prod-
uct no. N3520), was prepared in a 1:1 volumetric ratio. The
composition of both media can be obtained from the supplier. The
medium was supplemented with 2.5% (vol/vol) fetal bovine serum
(Sigma-Aldrich, product no. D5671) and 4 mM L-glutamine (Sigma-
Aldrich, product no. G7513).
ZnO NPs in powder form were purchased from Sigma-Aldrich
(product no. 544906) and suspended in deionized water to obtain a
stock suspension with nominal concentration of 10 g L−1 ZnO (cor-
responding to approximately 8 g L−1 Zn). The stock suspension was
sonicated in ultrasonic water bath for 30 min (Sonis 2 GT, Iskra PIO,
Slovenia) in order to break the NP aggregates and/or agglomerates.
The sonicated stock suspension was then diluted in the cell cul-
ture medium to obtain the final nominal concentration of 5 mg L−1
ZnO, which corresponds to approximately 4 mg L−1 Zn. The ZnO NP-
containing cell culture medium was kept in a cell culture incubator
at controlled atmosphere of 37 ◦ C, 5% CO2 , and 95% relative humid-
ity for 24 h. After the incubation, 8 mL aliquots of the cell culture
media were withdrawn from the incubator and ultracentrifuged
for 30 min at 100,000 g and 20 ◦ C (Beckman Coulter L8–70 M class
H preparative ultracentrifuge with Type 70.1 Ti rotor and 10 mL
thickwall polyallomer tubes). The supernatant from each sample
was further divided into two aliquots. Then 750 ␮L volumes were
withdrawn from the first supernatant aliquots and acidified with
750 ␮L of 65% HNO3 (Fischer Scientific, Leicester, UK). The acidified
supernatant aliquots were subjected to closed-vessel microwave
digestion in the Milestone Start D microwave lab station (Milestone,
Bergamo, Italy) equipped with the SK-10 high pressure segmented
rotor and 3 mL quartz microsampling inserts. The digestion was
conducted at 200 ◦ C and 700 W power, with step 1 (heating) for
15 min, and step 2 (constant temperature) for 15 min, followed
by 45 min cooling to 60 ◦ C. The total zinc content in the digests
was measured by flame atomic absorption spectroscopy (AAS;
PerkinElmer AAnalyst 100, Waltham, Massachusetts, USA). The sec-
ond supernatant aliquots, which were left unacidified, were used
for Zn(II) determination with the newly developed anodic stripping
voltammetric protocol using BiFE.
2.3. Apparatus
The ASV measurements were carried out using a modular
electrochemical workstation Autolab PGSTAT 10 (Eco Chemie,
Utrecht, Netherlands) in combination with the GPES 4.9 software
(Eco Chemie B.V.). The usual three-electrode configuration was
employed, i.e. a working bismuth film electrode (BiFE) prepared
in-situ on a supporting glassy carbon electrode (GCE, d = 2 mm),
a double-junction Ag/AgCl/KCl(satd.) reference electrode contain-
ing 0.1 mol L−1 HNO3 as the outer electrolyte, and a platinum
wire as the counter electrode. A computer-controlled magnetic
stirrer rotating at approximately 300 rpm was employed during
electrochemical accumulation and cleaning steps. All experiments
were carried out at room temperature of 23 ± 1 ◦ C in a 20 mL one-
compartment voltammetric cell.
2.4. Measurement procedures
The ASV measurements were conducted in the presence of dis-
solved oxygen. Following an electrochemical accumulation step
of usually 120 s at either −1.5 V or −1.6 V, and subsequent equi-
libration period of 15 s, the anodic stripping voltammogram was
recorded in the quiescent solution by applying a positive-going
square-wave potential scan from −1.5 V or −1.6 V to +0.4 V with
a frequency of 25 Hz, a potential step of 4 mV, and an amplitude of
50 mV. Before each measurement, a cleaning step was carried out
by applying a potential of +0.3 V for 30 s.
3. Results and discussion
To investigate the applicability of BiFE under less acid condi-
tions for measuring Zn(II), first the electroanalytical performances
of the unmodified glassy carbon electrode (GCE), the in-situ pre-
pared BiFE (both in 0.1 mol L−1 PIPES buffer solution containing
0.1 mol L−1 KNO3 with pH 6.5), and the in-situ prepared BiFE in
0.1 mol L−1 acetate buffer solution with pH 4.5 were compared. As
can be seen from Fig. 1, both BiFEs exhibited improved performance
in comparison to bare GCE with well-developed and narrower ASV
signals of Zn(II) at ca. −1.2 V. Additionally, the BiFE operating in
PIPES buffer solution revealed enhanced signal of Zn(II) for ca.
15% in comparison to that obtained in the acetate buffer solution,
together with a slight shift of the peak potential toward more neg-
ative values and significantly improved background current due to
attenuated hydrogen reduction reaction as a consequence of higher
pH of PIPES.
 T. Romih et al. / Sensors and Actuators B 238 (2017) 1277–1282 1279

Fig. 1. ASVs for four successive additions of Zn(II) in 10 ␮g L−1 steps together with background responses (dotted line) obtained at the unmodified GCE (a), at the in-situ
prepared BiFE (with 0.5 mg L−1 Bi(III)) in 0.1 mol L−1 PIPES buffer solution containing 0.1 mol L−1 KNO3 with pH 6.5 (b), and at the in-situ prepared BiFE (with 0.5 mg L−1 Bi(III))
in 0.1 mol L−1 acetate buffer solution with pH 4.5 (c); accumulation at −1.4 V for 120 s equilibration step of 15 s V, square-wave anodic stripping voltammetric scan with a
frequency of 25 Hz, an amplitude of 50 mV and a potential step of 4 mV; cleaning step of 30 s at +0.4 V.

Aimed at providing improved electroanalytical performance of
BiFE at pH 6.5, several key operational parameters were examined
and optimized. We investigated the effect of Bi(III) concentration
in the measurement solution upon ASV performance of the in-situ
prepared BiFE in the concentration range of 250 to 1000 mg L−1
Bi(III). The concentration of 500 ␮g L−1 yielded the highest signal
of Zn(II), thus we used this concentration in all further studies.
It is important to note that also other investigated concentra-
tions of Bi(III) resulted in similar, well-reproducible, but slightly
lower ASV signals. Fig. 2 shows the effect of the accumulation
potential (a) and accumulation time (b) upon the ASV signal of
20 ␮g L−1 Zn(II) recorded at BiFE in PIPES buffer solution contain-
ing 0.1 mol L−1 KNO3 and 500 ␮g L−1 Bi(III). As can be seen, the
ASV signals increased with the accumulation potential changing
from −1.3 V to −1.5 V and then started to decrease, apparently due
to commencement of a hydrogen evolution reaction. Thus, −1.5 V
was selected as the most favorable accumulation potential, which
is slightly more negative than the one usually employed in the
acetate buffer solutions, i.e. −1.4 V [2,3]. On the other hand, a step-
wise increase of the accumulation time resulted in an almost linear
increase of the 20 mg L−1 Zn(II) ASV signals up to 240 s, and at
prolonged accumulation times the signals started to level off as
a consequence of the electrode surface saturation. Therefore, the
accumulation time of 120 s was chosen for further studies as a
reasonable compromise between the signal height and the mea-
surement time.
Accordingly, Fig. 3 shows the anodic stripping voltammograms
for increasing concentrations of Zn(II) in the examined concen-
tration range of 10–100 ␮g L−1 associated with the accumulation
potential of −1.5 V for 120 s. Under these conditions, the BiFE exhib-
ited an excellent linear behavior with r2 of 0.994, surprisingly low
limit of detection (using 3 ␴ criterion) of 0.14 ␮g L−1 , and RSD of

Fig. 2. The effect of the accumulation potential (a) and accumulation time (b) upon the ASV signal of 20 ␮g L−1 Zn(II) at the in-situ prepared BiFE in 0.1 mol L−1 PIPES containing
0.1 mol L−1 KNO3 and 0.5 mg L−1 Bi(III) with pH 6.5; accumulation time of 60 s (a), accumulation potential of −1.5 V (b), and in the presence of 100 ␮L cell culture medium (c,
d); accumulation time of 120 s (c), accumulation potential of −1.6 V (d). Other conditions are as in Fig. 1.
1280 T. Romih et al. / Sensors and Actuators B 238 (2017) 1277–1282
Fig. 3. ASVs for ten successive additions of Zn(II) in 10 ␮g L−1 steps together with background response (dotted line) obtained at the in-situ prepared BiFE after 120 s
accumulation at −1.5 V. Inset shows the corresponding calibration plot. Other conditions are as in Fig. 2.
1.7% (20 ␮g L−1 , n = 12). These characteristics clearly corroborate
the aptness of BiFE for measuring low concentration levels of Zn(II)
in less acidic solutions. In addition, the Zn(II) signals were shifting
slightly toward more positive potentials with increasing Zn(II) con-
centration reflecting the attractive forces between deposited metal
particles [11].
Since the in-situ prepared BiFE exhibited promising operation
in the PIPES buffer solution, further studies were carried out to
develop a convenient protocol for measuring Zn(II) in a more com-
plex organic matrix, i.e. in the cell culture medium (at this stage
without ZnO NPs). We employed the in-situ preparation protocol to
provide a fresh electrode surface after each measurement together
with the standard addition method to diminish the effect of possi-
ble interferent(s). The total background concentration of Zn(II) as
a micronutrient in the undiluted cell culture medium was deter-
mined by flame atomic absorption spectroscopy to be 8 ␮g L−1 ,
which falls below the limit of detection of the present method when
using a 200-fold dilution in the supporting PIPES buffer solution,
i.e. being ca. 0.04 ␮g L−1 Zn(II); therefore, it should not contribute
to false positive signals. Upon the addition of 100 ␮L cell culture
medium, the signal of 20 ␮g L−1 Zn(II) associated with the accumu-
lation of 120 s at −1.5 V was attenuated for ca. 64%; however, due
to very favorable sensitivity (and repeatability) of the proposed
method, low concentrations of Zn(II) could still be readily mea-
sured; thus, further optimization and investigation were carried
out under these conditions.
Fig. 2 depicts also the influence of the accumulation potential (c)
and the accumulation time (d) upon the signal of 20 ␮g L−1 Zn(II) in
the presence of 100 ␮L cell culture medium in 20 mL PIPES buffer
solution. In this case, the most favorable accumulation potential
for measuring Zn(II) was −1.6 V, i.e. instead −1.5 V in the absence
of cell culture medium. The signal did not level off even at the
prolonged accumulation times up to 360 s, implying on a thin mem-
brane/barrier formation at the electrode surface which, however,
still allowed convenient measurements of low concentration levels
of Zn(II).
The electroanalytical performance of BiFE was further inves-
tigated in the presence of 100 ␮L cell culture medium in 20 mL
PIPES buffer solution under adopted conditions. Fig. 4 depicts the
anodic stripping voltammograms for increasing concentration of
Zn(II) in the examined range of 10–100 ␮g L−1 in combination with
120 s accumulation. Also in this case the Zn(II) signals were well-
developed over low background, and, similarly as without the cell
culture medium, the Zn(II) signals were shifting towards slightly
less negative potentials with increasing concentrations of Zn(II)
exhibiting the same stripping pattern. Note that in both cases, i.e.
in the absence and in the presence of cell culture medium, the
stripping/re-oxidation signal of bismuth increased with increas-
ing concentration of Zn(II), and very interestingly, it was observed
that the bismuth stripping/re-oxidation signal was not signifi-
cantly affected by the presence of cell culture medium. This reveals
another attractive characteristic, i.e. the robustness of BiFE used for
this purpose.
Under these conditions the BiFE exhibited surprisingly good lin-
ear behavior with r2 of 0.999, favorable limit of detection (using
3 ␴ criterion) of 0.15 ␮g L−1 and RSD of 3.0% (20 ␮g L−1 , n = 12). This
demonstrates that, apart from the decreased Zn(II) signal, the over-
all performance of the BiFE electrode did not change significantly
in the presence of potentially interfering organic species, i.e. the
constituents of cell culture medium; however, and in accordance
with Fig. 2d, the stripping signal can be considerably enhanced,
if necessary, via corresponding prolongation of the accumulation
time.
Finally, the electroanalytical performance of the BiFE was
investigated through measurements in the cell culture medium
containing also 5 mg L−1 of ZnO NPs (corresponding to approxi-
mately 4 mg L−1 of Zn) that had been incubated at 37 ◦ C for 24 h,
which is a common protocol in cell biology experiments. Before
the measurements, the NP-containing cell culture medium was
ultracentrifuged to sediment the ZnO NPs and therefore mini-
mize the possibility of their interference when measuring Zn(II).
The flame atomic absorption spectrometry revealed that the total
Zn content in the original cell culture medium containing ZnO
NPs was 3.94 ± 0.07 mg L−1 (n = 2), whereas its supernatant con-
tained 2.90 ± 0.05 mg L−1 Zn in total (n = 2). Also in accordance with
another report on ZnO NPs in cell culture medium [12], our results
indicate that the NP sedimentation was inefficient despite the
stringent ultracentrifugation conditions employed, i.e. 100,000 g
for 30 min. This underlines the convenience of anodic stripping
voltammetry for estimating the amount of Zn(II) dissolved from
ZnO NPs in complex matrices of organic molecules. In addition, the
ZnO powder used in the present study was composed of particles
 T. Romih et al. / Sensors and Actuators B 238 (2017) 1277–1282 1281

Fig. 4. ASVs for ten successive additions of Zn(II) in 10 ␮g L−1 steps together with background response (dotted line) in the presence of 100 ␮L cell culture medium obtained
at the in-situ prepared BiFE after 120 s accumulation at −1.6 V. Inset shows the corresponding calibration plot. Other conditions are as in Fig. 2.

Fig. 5. ASVs recorded in the ultracentrifuged cell culture medium containing ZnO NPs (dashed line), together with three consecutive standard additions of 10 ␮g L−1 Zn(II)
(full lines), and the background response (dotted line); cleaning step of 120 s at +0.4 V. Inset shows the corresponding standard addition plot. Other conditions are as in Fig. 4.

ranging from tens of nanometers to several hundred nanometers in extrapolation and with respect to the abovementioned dilution)
diameter [13]; it has been shown that NPs > 5 nm should not con- 1.9 ± 0.3 mg L−1 (n = 2), i.e. ca. 48% of the ZnO NPs were dissolved.
tribute to the net signal increase when measuring metal ions in the
presence of ZnO NPs [9], and as has been already demonstrated for
Ag NPs in our recent study [14].
4. Conclusions
Considering the expected content of Zn(II) in the real sample,
150 ␮L of cell culture medium was added into 20 mL of 0.1 mol L−1
We have demonstrated a straightforward method comprising
PIPES buffer solution containing 0.1 mol L−1 KNO3 and 0.5 mg L−1
the in-situ prepared BiFE that allows expedient anodic stripping
Bi(III). Since the preliminary experiments unveiled a slight memory
voltammetric measurements of low concentrations of Zn(II) in less
effect in this case, the cleaning step was prolonged to 120 s at +0.4 V.
acidic medium, i.e. in 0.1 mol L−1 PIPES buffer solution with pH 6.5
The protocol for the determination of dissolved Zn(II) comprised
(approaching circumneutral pH), thus providing extended scope
a standard addition method with three consecutive additions of
and applicability of the BiFE in addition to the hitherto typically
10 ␮g L−1 Zn(II). As depicted in Fig. 5, an excellent linear depen-
employed acetate buffer solution of pH around 4.5. The attrac-
dence was achieved with a correlation coefficient r2 of 0.9997
tive application of the proposed protocol was successfully tested
and the corresponding concentration of Zn(II) was (evaluated by
through measuring Zn(II) in the presence of complex organic matrix
1282 T. Romih et al. / Sensors and Actuators B 238 (2017) 1277–1282
of cell culture medium containing unsedimented ZnO nanoparticles
for the purpose of nanotoxicological study.
Acknowledgments
The work was supported by the Slovenian Research Agency
(Grant No. P1-0034) and the NanoFASE project (Grant Agree-
ment No. 6460020) within the framework of the EU Horizon 2020
research and innovation programme. Dr. Tea Romih acknowledges
the study grant entitled “Innovative scheme of co-funding doctoral
studies for promoting co-operation with the economy and solving
contemporary social challenges” (Grant No. 1291), provided jointly
by the Ministry of Education, Science and Sport of the Republic of
Slovenia and the EU Social Fund. Veno Kononenko acknowledges
funding by the Slovenian Research Agency (Grant No. 1000-14-
0510).
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