Journal of Electroanalytical Chemistry 775 (2016) 72–76

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Journal of Electroanalytical Chemistry

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

Development of a tetraphenylporphyrin cobalt (II) modified glassy

carbon electrode to monitor oxygen consumption in biological samples
L.P.H. Saravia a, S. Anandhakumar a,⁎, A.L.A. Parussulo a, T.A. Matias a, C.C. Caldeira da Silva b, A.J. Kowaltowski b,
K. Araki a, M. Bertotti a,⁎
a
Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo
b
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av Prof. Lineu Prestes, 748 São Paulo, SP, Brazil

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

Article history: A glassy carbon (GC) electrode modified with a CoTPP (5, 10, 15, 20-meso tetraphenylporphyrin cobalt (II)) film
Received 25 February 2016 was prepared and used as a dissolved oxygen electrochemical sensor. The as-prepared electrode was character-
Received in revised form 29 April 2016 ized using atomic force microscopy (AFM) and cyclic voltammetry (CV). The electrochemical behaviour of the
Accepted 18 May 2016
CoTPP/GC modified electrode for oxygen reduction in 0.1 mol L−1 KNO3 aqueous solution was compared to
Available online 19 May 2016
that of a bare GC electrode and a decrease in overpotential (~0.30 V) and onset potential (~0.15 V) was noticed.
Keywords:
The proposed platform was used as an electrochemical sensor for dissolved oxygen detection, and analytical pa-
Dissolved oxygen sensor rameters such as reproducibility, repeatability and long-term stability were determined. The usefulness of the de-
CoTPP veloped sensor was demonstrated by continuous amperometric monitoring of mitochondrial oxygen
Amperometry consumption and the results agreed well with those obtained in parallel measurements performed using a com-
Mitochondrial respiratory cycle mercial high-resolution “Clark-type” electrode.
Electrochemical sensor © 2016 Elsevier B.V. All rights reserved.

1. Introduction extensive research has been performed to develop alternative materials

The development of economically viable sensors and/or devices to
get real-time information on dissolved oxygen concentration in envi-
ronmental, clinical and biological samples is one of the most active re-
search areas in electroanalytical chemistry [1–3]. The well-known
commercially available Clark electrode is widely used for this purpose
[4–7]. Despite its popularity, several drawbacks limit its application
e.g. (i) the resultant current is particularly unstable and therefore regu-
lar pre-calibrations are required, which is not convenient for continuous
monitoring, especially in biological systems; (ii) even though platinum
and gold are inert for surface sensitive reactions, from the commercial
point of view the cost of these materials makes them undesirable [8,9].
Conventional titrations and instrumental methods based on colori-
metric, electrochemical (including potentiometry, amperometry, and
voltammetry), fluorescence and chemiluminescence information have
also been used [10–18]. However, electrochemical methods are most
widely used due to their simplicity, sensitivity, selectivity, ease of oper-
ation and low cost [19]. The oxygen reduction reaction in solid elec-
trodes usually depends on high overpotential because of its slow
kinetics [20,21]. Platinum-based catalysts are used mostly due to their
high catalytic activity, but the cost of this metal has bolstered attention
towards finding alternative, platinum-free, catalysts [22–24]. Therefore,
⁎ Corresponding authors.
E-mail addresses: anandchemist@gmail.com, mbertott@iq.usp.br (M. Bertotti).
and/or catalysts with high electrocatalytic activity for the electrochem-
ical reduction of oxygen. Cobalt phthalocyanine was first reported as an
excellent catalyst for oxygen reduction [25] and different mediators
were then reported, such as DNA [26], antraquinone [27,28],
polymethylene blue [29], peptide nanostructure [30], cobalt complexes
(cobalt (II) porphyrins, cobalt phthalocyanines etc.) [31–37], iron (II)
tetrasulfonated phthalocyanine and iron (II) tetra-(N-methyl pridyl)-
porphyrin multilayers [21], magnesium (II) phthalocyanine [38],
nickel-salen polymeric film [39], vitamin B-12 [40], ABTS-laccase [41],
sub-micron ZnO doped RuO2 [42], diamino-o-benzoquinone [43],
indigotetrasulfonate [44] and eugenol [45].
To extend our research interest to dissolved oxygen sensing, we
have chosen cobalt (II) tetraphenylporphyrin as a mediator attached
to a glassy carbon support. The kinetics of the electrode process and
the possibility of using the proposed platform for real-time monitoring
of dissolved oxygen have been investigated in this work.
2. Experimental methods
2.1. Reagents and apparatus
Analytically pure chemicals were used in this work and the aqueous
solutions were prepared using 18.2 MΩ cm resistivity water (Millipore).
Nafion, potassium nitrate (KNO3), potassium chloride (KCl), HEPES (4-
(2-hydroxyethyl)-1-piperazineethanesulfonic acid), phosphate, MgCl2,

http://dx.doi.org/10.1016/j.jelechem.2016.05.026
1572-6657/© 2016 Elsevier B.V. All rights reserved.
 L.P.H. Saravia et al. / Journal of Electroanalytical Chemistry 775 (2016) 72–76
succinate, ADP and oligomycin were purchased from Sigma Aldrich and
used as received. Cyclic voltammetry and amperometry experiments
were carried out using a PalmSens portable Potentiostat (Netherland).
A three electrode electrochemical cell containing a CoTPP/GC modified
electrode (3 mm diameter), a Ag/AgCl (saturated KCl) and a platinum
foil served as working, reference and counter electrodes, respectively.
All measurements were performed at room temperature (25 ± 1 °C).
Atomic force microscopy (AFM) images were obtained using a
PicoSPM-LE Molecular Imaging System, slightly below their resonance
frequency of approximately 294 kHz in air with cantilevers operating
in the intermittent-contact mode (AAC mode) to measure the topogra-
phy of the surface.
2.2. Synthesis of CoTPP
TPP (meso-tetraphenylporphyrin) (0.0594 g) was dissolved in 9 mL
of DMF and heated at 145 °C in a silicone bath for 20 min. Subsequently,
CoCl2·6H2O (0.0596 g) was added into the solution and the reaction
was continued for another 20 min (145–150 °C). After the reaction,
the solution was cooled at room temperature and kept in a refrigerator
for 12 h. Finally, the reaction product (CoTTP) was filtered, dried and
used for the modification of the GC surface.
2.3. Preparation of the CoTPP/GC electrode
The GC electrode was well polished with a polishing cloth using
0.05 μm alumina powder and sonicated for 5 min. The synthesized
CoTPP powder (1 mg mL−1) was dispersed in chloroform and Nafion
(0.1%) solution and sonicated for 10–15 min to form a homogeneous
suspension. Afterwards, 10 μL of this solution were drop casted on the
GC surface and then allowed to dry in air.
2.4. Preparation of standard oxygen solutions
The saturated dissolved oxygen solution (1.24 × 10−3 mol L−1) [46]
was prepared by passing the oxygen gas into a 0.1 mol L−1 KNO3 solu-
tion. Solutions with known oxygen concentrations were prepared by
transferring fixed amounts of the oxygen saturated solution to the elec-
trochemical cell completely filled with supporting electrolyte solution,
which initially contained no oxygen. Scheme 1 shows the proposed ap-
paratus, which was adapted from the literature [3]. The concentration of
oxygen in the electrochemical cell after each addition of the saturated
oxygen solution can be calculated as described in [3].
Scheme 1. Oxygen calibration system design. A: oxygen cylinder, B: oxygen saturated solution, C: valve, D: piston burette, E: counter electrode, F: working electrode, G: reference
electrode, H: solution out, I: electrochemical cell, J: magnetic stirrer.
73
2.5. Mitochondrial isolation and incubation
All experiments were approved by the local Animal Care and Use
Committee and follow international NIH-like vertebrate research proto-
cols. Mitochondria were isolated from adult mouse livers using standard
differential centrifugation protocols described in [47] and kept over ice
until the experiments were conducted. Oxygen consumption traces
were obtained by incubating the isolated mitochondria in a 37 °C
media composed of 125 mmol L− 1 sucrose, 65 mmol L− 1 KCl,
10 mmol L−1 HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid), 2 mmol L−1 Na₂HPO₄ and 4 mmol L−1 MgCl2. The pH was adjusted
to 7.2 using KOH.
3. Results and discussion
3.1. Microscopic characterization of CoTPP
The prepared CoTPP dispersed solution was coated on a mica surface
for atomic force microscopy analysis (AFM) and the obtained results are
given in Fig S1. From the AFM images, it is clearly seen that CoTPP par-
ticles have a rod-like shape with aspect ratio (~0.4:0.1 μm) of ~4 and
were uniformly coated on the surface. The edges of the rod-like struc-
tures may have more active sites which can accelerate the electron
transfer process involving oxygen reduction on the CoTPP electrode
surface.
3.2. Electrochemical characterization of the CoTPP/GC modified electrode
Cyclic voltammograms recorded with the CoTPP/GC modified elec-
trode in a 0.1 mol L− 1 KNO3 solution at different scan rates (0.01–
0.15 V s− 1) are shown in Fig. 1A. The well-defined redox couple ob-
served at around 0.2 V corresponds to the CoII/CoIII electron transfer
process. The oxidation and reduction peak current values increase line-
arly with the scan rate (Fig. 1B), demonstrating that this is a typical sur-
face-controlled electrode process. In order to calculate the surface
coverage (Г) of the CoTPP film, the slope obtained from the anodic cal-
ibration plot (Fig.1B) was used in the equation Г = ip4RT/n2F2Aυ [39],
where ip is the anodic peak current, R is the universal gas constant
(8.314 J mol−1 K), T is the absolute temperature (298 K), n is the num-
ber of electrons transferred in the redox process, F is the Faraday con-
stant (96,485 C mol−1), A is the electrode area (0.07065 cm2) and υ is
the scan rate (V s−1). The calculated surface coverage for the CoTPP/
GC modified electrode was found to be 4.75 × 10−11 mol cm−2.
74 L.P.H. Saravia et al. / Journal of Electroanalytical Chemistry 775 (2016) 72–76
Fig. 1. Cyclic voltammograms recorded with a CoTPP/GC modified electrode in 0.1 mol L−1 KNO3 solution at different scan rates (A); corresponding ip vs υ plot (B).
3.3. Electrocatalytic reduction of dissolved oxygen at the CoTPP/GC modified
electrode
Typical cyclic voltammograms of dissolved oxygen in 0.1 mol L−1
KNO3 solution (pH 7) using GC and CoTPP/GC modified electrodes at
the scan rate of 0.1 V s−1 are shown in Fig. 2. The oxygen reduction
peak is located at a potential of − 0.72 V for the bare GC electrode,
whereas the same process takes place at − 0.42 V for the CoTPP/GC
modified electrode which is comparable to reported values for various
modified electrodes (Table S1). By comparing both voltammograms,
the overpotential for the electroreduction of oxygen can be clearly
seen to be shifted towards a less negative potential value (about
0.30 V) when the experiment was carried out with the modified elec-
trode. Furthermore, it is also interesting to compare onset potential
values (the less negative potential at which a reaction product is
formed, i.e., the raising part of a cyclic voltammogram) of bare GC and
CoTPP/GC electrodes, which are −0.30 V and −0.15 V for the GC and
the CoTPP/GC electrodes, respectively. Therefore, in addition to the de-
crease in the overpotential, the onset potential was also significantly
shifted towards a less negative potential value. The results obtained
confirm the electrocatalytic behaviour of the CoTPP/GC. This can be rea-
soned as (i) dioxygen binding to cobalt(II) axial position facilitates the
bielectronic reduction reaction; (ii) coordination to Co(II)TPP changes
dioxygen energy levels shifting the redox potential to less negative po-
tentials and (iii) the electrocatalytic activity of cobalt center is modulat-
ed by the porphyrin ring [34].
To understand the kinetics of the overall electron transfer process in-
volving the oxygen reduction at the CoTPP/GC modified electrode, cyclic
voltammograms were recorded at various scan rates from 0.02 to
Fig. 2. Cyclic voltammograms of dissolved oxygen (saturated) in a 0.1 mol L−1 KNO3
solution recorded with a GC (b) and a CoTPP/GC modified electrode (c). Curve (a)
corresponds to the voltammogram of supporting electrolyte in absence of oxygen
recorded with a GC electrode. Scan rate: 0.1 V s−1.
0.2 V s− 1 in 0.1 mol L−1 KNO3 aqueous neutral solution containing
100% dissolved oxygen (Fig. S2A). The oxygen reduction peak current
increases with scan rate and the linear dependence of peak current
with the square root of scan rate (Fig. S2B) indicates the electrode pro-
cess is mass-transport controlled. Peak current and scan rate values
were also plotted in logarithmic scale, and a slope value of ~ 0.44 was
obtained. This value is close to that expected (0.5) for a process con-
trolled by diffusion.
Further, in order to get information on the mechanism of oxygen re-
duction at the CoTPP/GC modified electrode, i.e. whether oxygen is re-
duced through a two electron pathway (formation of hydrogen
peroxide) or a direct four electron pathway (formation of water), the
transfer coefficient (α) and the number of electrons involved in the
rate determining step (nc) were calculated from the peak potential
(Epc) versus log scan rate plot (Fig. S2C). From the slope value
(~ 0.054 V) of this plot, the Tafel slope (b) was calculated (from the
equation Ep = b/2 log υ + constant) as 0.108 V. The values of α and nc
were calculated using the Tafel slope (from the equation y = 2.303RT/
αncF, where y = Tafel slope and the other parameters have standard
meaning) [48,49] and were found to be 0.54 and 1.09, respectively.
Afterwards, the total number of electrons (n) involved in the oxygen
reduction reaction was calculated from the slope value (120.6 μA)
obtained in Fig. S2B using the equation for a diffusion controlled
irreversible process ip = 2.99 × 105ACo⁎D1/2 o υ
1/2
n (αnc)1/2 [49] where
ip = peak current (A), A = electrode area (0.07065 cm2), Co⁎ = concentra-
tion of dissolved oxygen (saturated solution = 1.24 × 10−6 mol cm−3),
D o = diffusion coefficient of oxygen in aqueous medium
(1.9 × 10−5 cm2 s− 1) [21], υ = scan rate (V s− 1), n = total number
of electrons, α = transfer coefficient (0.54), and nc = number of elec-
trons involved in the rate determining step). The n value was found to
be ~1.44, which suggests that the oxygen reduction follows a two elec-
tron pathway, where hydrogen peroxide is the main product and the
transfer of one electron is the rate determining step at the CoTPP/GC
modified electrode.
3.4. Analytical applications
The analytical performance of the CoTPP/GC modified electrode was
evaluated by varying the concentration of dissolved oxygen in the elec-
trochemical cell according to a procedure proposed in the literature and
shown in Scheme 1 [3]. Cyclic voltammetric responses in 0.1 mol L−1
KNO3 solution are presented in Fig. 3A and the resulting current mea-
sured at −0.42 V increases linearly as the dissolved oxygen concentra-
tion is increased in the range of 5 to 48.7% (62 to 604 × 10−6 mol L−1,
correlation coefficient of 0.9920, Fig. 3B). From the slope value of the
calibration plot, the limit of detection (LOD = 3Sb/S, where, Sb = stan-
dard deviation of the blank; S = slope of the analytical curve) [38] was
calculated and found to be 0.035% (0.4 × 10−6 mol L−1), which is rea-
sonably comparable to some reported values (Table S1). The other
 L.P.H. Saravia et al. / Journal of Electroanalytical Chemistry 775 (2016) 72–76
Fig. 3. Cyclic voltammograms recorded at various concentrations of dissolved oxygen (5–48.7%) in 0.1 mol L−1 KNO3 solution using the CoTPP/GC modified electrode (A). Corresponding
calibration plot of current (measured at −0.42 V) versus oxygen concentration (B). Scan rate = 0.1 V s−1.
important analytical parameters such as reproducibility, repeatability
and long-term stability were evaluated by cyclic voltammetry. The re-
producibility studies were carried out using five different CoTPP/GC
modified electrodes and the repeatability measurements were per-
formed with one modified electrode at different interval times (n =
10). Relative standard deviation (RSD) values were found to be 2.6
and 2.7%, respectively. Long-term stability was assessed by measuring
the oxygen response for five consecutive days using the same modified
electrode and the calculated RSD was found to be 2.6% (between mea-
surements, the electrode was stored at ambient temperature).
In order to evaluate the practical application of the proposed sensing
platform for continuous monitoring of dissolved oxygen in aqueous
neutral solutions, amperometric measurements at an applied potential
of −0.5 V were performed. The experiment was designed to vary oxy-
gen concentration by bubbling oxygen and argon repeatedly. Fig. 4
shows the result of such experiment and the current increase (during
O2 bubbling) and further decrease (during argon bubbling) clearly dem-
onstrate that the sensor responds rapidly to changes in the dissolved ox-
ygen concentration.
Finally, the possibility of using the proposed sensor to follow respira-
tory activity in a biological system was evaluated by using this electrode
to monitor oxygen consumption in isolated liver mitochondria. The re-
sponse towards oxygen was continuously measured using the proposed
electrochemical sensor (Fig. 5, blue trace) and parallel measurements
were performed with a high-resolution Clark electrode Oroboros sys-
tem (Fig. 5, red trace), an equipment considered the gold standard sys-
tem for oxygen consumption detection in liquid biological systems [50].
A slow decrease in oxygen concentrations was detected by both systems
in the presence of mitochondria (additions a, a′). The subsequent addi-
tion of succinate (b, b′), which acts as a substrate for mitochondrial elec-
tron transport to oxygen, substantially increased oxygen consumption
Fig. 4. Amperometric response measured with the CoTPP/GC modified electrode in
0.1 mol L−1 KNO3 solution upon bubbling of oxygen and argon. E = −0.5 V.
75
rates detected by both systems. ADP (c, c′) further increased this con-
sumption by activating mitochondrial oxidative phosphorylation [47].
Finally, the addition of oligomycin blunted the ADP-induced increase
in oxygen consumption due to its inhibitory effect on mitochondrial ox-
idative phosphorylation (d, d′). All measurements were very similar
using both electrodes.
The similarity in detection speed and range to a high-resolution
commercial electrode confirms the usefulness of the proposed modified
electrode as an amperometric sensor for oxygen in biological samples.
4. Conclusions
We have successfully prepared a simple and effective dissolved oxy-
gen sensor based on deposition of a CoTPP film onto a GC electrode. Ki-
netic studies were carried out systematically and the results suggested
the electroreduction of oxygen at the CoTPP/GC modified electrode is
a 2-electron process, i.e. hydrogen peroxide is the main product. The
usefulness of the proposed sensor for real time oxygen monitoring
was demonstrated by continuous measurements of the concentration
of the target analyte in aqueous solution as well as in a biological
samples.
Further studies are under progress to use such a sensor as a tip in
SECM (Scanning Electrochemical Microscopy), a technique with several
advantages for studying live cells, cellular processes and cell response
towards drug administration. Localized microelectrochemical informa-
tion on oxygen uptake by a single cell can be obtained by scanning the
Fig. 5. Amperometric response of the CoTPP/GC modified electrode (E = −0.5 V) and
oxygen concentration measured with a Clark electrode in cellular medium upon
addition of mitochondria (a, a′, ~0.5 mg mL−1), succinate (b, b′ 1 mmol. L−1), ADP (c, c′,
1 mmol L−1) and oligomycin (d, d′, 1 μg mL−1).
76 L.P.H. Saravia et al. / Journal of Electroanalytical Chemistry 775 (2016) 72–76
tip in close proximity across the cell and this information can be used as
an indicator of respiratory activity.
Acknowledgements
The authors thank FAPESP grants 13/7937-8, 10/51906-1, 12/20676-6,
CNPq grant 471236/2013-6 and CAPES for financial support and SAK
gratefully acknowledges the Sao Paulo Research Foundation (FAPESP)
for the Postdoctoral Fellowship grant [14/15215-5].
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jelechem.2016.05.026.
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