Journal of The Electrochemical Society, 163 (9) B499-B506 (2016) B499

0013-4651/2016/163(9)/B499/8/$33.00 © The Electrochemical Society

Development of an Electrochemical Sensor Based on LbL Films

of Pt Nanoparticles and Humic Acid
Monalisa dos Santos,a Ellen Christine Wrobel,a Vagner dos Santos,b Sueli Pércio Quináia,c
Sérgio Toshio Fujiwara,a Jarem Raul Garcia,a,∗ Christiana Andrade Pessôa,a
Elizabeth W. O. Scheffer,a and Karen Wohnratha,∗,z
a Departamento de Quı́mica, Universidade Estadual de Ponta Grossa (UEPG), Ponta Grossa, Paraná 84030-900, Brazil
b Instituto Federal de Educação, Ciência e Tecnologia do Paraná (IFPR), Curitiba, Paraná 80230-150, Brazil
c Departamento de Quı́mica, Universidade Estadual do Centro-Oeste (UNICENTRO), Guarapuava,
Paraná 85040-080, Brazil

We report the first attempt to use a naturally occurring biopolymer, such as humic acid (HA), as modifiers to prepare a new organic-
inorganic composite film with the 3-n-propylpyridiniumsilsesquioxane chloride Pt nanoparticles (Pt-SiPy+ Cl− ). By using layer-
by-layer (LbL) technique, we have constructed multilayered films, (HA/Pt-SiPy+ Cl− )n and (Pt-SiPy+ Cl− /HA)n , with adjustable
thickness due to negatively charged macromolecule HA, which assists the electrostatic interaction with the positively charged
of the Pt-SiPy+ Cl− nanohybrid. The combination of these partially charged polyelectrolytes in the LbL film was explored by
spectroscopic and electrochemical techniques. The formation of the film (HA/Pt-SiPy+ Cl− )n is more efficient, since the presence of
the macromolecule in the inner layer provides a greater number of anchoring sites for the hybrid, ensuring a more linear and uniform
growth of the LbL films. This film also presented a better electroanalytical response for oxidation of 17α-ethynylestradiol, EE2,
indicating that the Pt nanoparticles strongly influences the electrochemical oxidation of estrogen. The analytical curve for EE2 was
linear in the concentration range of 1.37 to 21.4 μmol L−1 (R = 0.998), with a detection limit of 1.10 μmol L−1 and quantification
limit of 3.68 μmol L−1 . The obtained results showed that the synergism between HA and Pt-SiPy+ Cl− improves the electroactive
properties of the sensor.
© 2016 The Electrochemical Society. [DOI: 10.1149/2.1001609jes] All rights reserved.

Manuscript submitted April 11, 2016; revised manuscript received June 27, 2016. Published July 22, 2016.

Humic acids (HA) are the most important components of hu-
mic substances,1 which are organic macromolecules obtained from
the microbiological decomposition of animals and plants.2–4 HA
derivatives are anionic polyelectrolyte compounds,5 which con-
tain a large number of carboxyl, carbonyl, hydroxyl, hydrophobic
aliphatic and aromatic groups.2 Due to their particular structure,
humic acids could act as modifiers6 in the preparation of chemi-
cally modified electrodes,5,7–11 which have been applied to detection
of heavy metals,5,7 nitrite,8 pentachlorophenol,9 polycyclic aromatic
hydrocarbons12 and herbicide.2
Humic acids have been also employed in layer-by-layer self-
assembly technique,9,13 which is based on electrostatic interactions
of positively and negatively charged polyelectrolytes and it provides
nanometer-thick films with high homogeneity,14 due to their polyelec-
trolytic characteristics. Galeska et al.13 studied the formation of LbL
films of HA and ferric ions, which were used as semi-permeable mem-
branes for an implantable glucose sensor. Crespilho et al.9 evaluated
the electrochemical sensor for pentachlorophenol pesticide, based on
LbL films of HA and poly(allylamine hydrochloride), with a detection
limit of 0.0016 μmol L−1 . The presence of humic acid in the LbL films
improves the adsorption of pentachlorophenol at the electrode surface.
In the last years, the development of multiple human activities,
like as industry, transport, agriculture and urbanization, has been gen-
erated adverse consequences to the environment and human health.15
Emerging pollutants are compounds without regulatory status and rep-
resent potential threats to ecosystems and human health because of
their toxic effects.16–18 Hormones have become important emerging
contaminants due to incomplete removal in wastewater treatment18
and because they can display a biological response at very low
concentrations.19 Hormones in wastewater cause adverse effects in
humans or wildlife.18 Therefore, several hormones must be controlled,
such as 17α-ethynylestradiol (EE2), 17α-estradiol, estrone (E1), 17β-
estradiol (E2) and estriol (E3). Several analytical techniques, includ-
ing high-performance liquid chromatography,20 spectrophotometry21
and gas chromatography-mass spectrometric,22 have been described
to the hormones detection, and from these techniques low detection
and quantitation limits were obtained (1.0–450.0 ng mL−1 ). In addi-
tion, electrochemical techniques for detection of hormones have been
∗ Electrochemical Society Member.
z
E-mail: karen.woh@gmail.com
found in the literature, as cyclic voltammetry,23 differential pulse
voltammetry24 and square wave voltammetry,25 which are simple,
inexpensive and short analysis time, when compared with different
experimental techniques.26
In order to improve the sensitivity and selectivity in the determi-
nation of estrogen hormones, chemically modified electrodes can be
an alternative to the conventional electrodes,27 since they are more
selective and assist the electron transfer process.28,29 Ma et al.27 de-
veloped an electrochemical sensor for detection of diethylstilboestrol
based on a glassy carbon electrode modified with graphene and gold
nanoparticles. From the cyclic voltammetric results, it was obtained
a detection limit of 0.0098 μmol L−1 . Yuan et al.30 evaluated the
electrochemical detection of 17β-estradiol using a molecular imprint-
ing polymer based on glassy carbon electrode modified with platinum
nanoparticles. From the differential pulse voltammetry, it was obtained
a detection limit of 0.016 μmol L−1 .
The ionic organosilane 3-n-propylpyridinium silsesquioxane poly-
mer, here named as SiPy+ Cl− , it is a polyelectrolyte which has strong
anionic exchange capacity31,32 and high solubility in water and appre-
ciable capacity to form stable thin films on solid matrix surfaces. Due
to these properties, the SiPy+ Cl− has been used as stabilizer on the
synthesis and incorporation of metallic nanoparticles, MNp-Sipy+ Cl−
(M = Pt, Ag and Au).32–35 These nanocomposites were obtained by re-
duction in situ of metal salt solutions with nanoparticles sizes around
6–10 nm. For this reason, this ionic hybrid consists as an interest-
ing material for the construction of LbL films. The presence of Pt
nanoparticles confined in a silsesquioxane matrix containing charged
group make them an interesting material for the construction of an
electrode for the detection of this emerging pollutant.
In this context, this study describes the development of an electrode
based on the organic-inorganic hybrid of Pt-SiPy+ Cl− (Pt nanopar-
ticles incorporated into the inorganic polymer 3-n-propylpyridinium
silsesquioxane polymer) and a natural organic polymer, such as the
humic acid. The multilayered film was used to electrochemical deter-
mination of 17α-ethynylestradiol.
Experimental
Chemicals and solutions.—The reagents used in this study were
of analytical grade and purchased from Sigma-Aldrich. Stock solu-
tion of 17α-ethynylestradiol (1.0 × 10−3 mol L−1 ) was prepared in

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 B500 Journal of The Electrochemical Society, 163 (9) B499-B506 (2016)

Figure 1. Schematic illustration of fabrication procedure of LbL film in the configuration HA/Pt-SiPy+ Cl− : The solid substrate was immersed in the humic acid
solution for 4 minutes (1). In the following step, the excess of molecules was removed by immersing the substrate in the washing solution at pH = 7.9 for 10
seconds (2). The substrate containing the HA layer was immersed in the Pt-SiPy+ Cl− solution for 4 minutes (3). So, the non-adsorbed molecules were removed
in the washing solution at pH = 2.9 for 10 seconds (4).

acetonitrile. Humic acid solution (0.5 g L−1 ) was solubilized in
pH = 9.0, to ensure complete dissolution of HA.9 The polymer
SiPy+ Cl− was synthesized according to the procedure described in
the literature.36,37
Different solutions were tested as supporting electrolytes (at 0.1
mol L−1 ): NaH2 PO4 (pH = 4.3); Na2 HPO4 (pH = 9.4); NaNO3 (pH =
6.2); KCl (pH = 6.5); phosphate buffer (pH = 7.5); phosphate buffered
saline (pH = 7.2) and Britton-Robinson buffer (pH = 2.0). All the
experiments were performed with doubly distilled water.
Synthesis of the Pt-SiPy+ Cl− nanohybrid.—The synthesis of the
platinum nanoparticles incorporated to the SiPy+ Cl− polymer was
performed according to the procedure related in the literature.32 This
method consists in the mixing of hexachloroplatinic acid (H2 PtCl6 )
(2 × 10−3 mol L−1 ), SiPy+ Cl− (0.5 g L−1 ) and formic acid (0.1 mol
L−1 ), which was used as the reducing agent, at proportion of 1:1:1
(v/v). The solution was mixed for 12 hours at room temperature under
magnetic stirring until the Pt-SiPy+ Cl− nanohybrid was obtained.
This synthesis was performed in dark environment, which prevents
ambient light interference.
Films assembly.—Pt-SiPy+ Cl− and humic acid were employed
as cationic and anionic polyelectrolytes, at pH = 2.9 and 7.9, re-
spectively. LbL films were prepared at two different configura-
tions: (HA/Pt-SiPy+ Cl− )n (nanohybrid as the last layer) and (Pt-
SiPy+ Cl− /HA)n (with humic acid as the last layer), where n is the
bilayers number. The LbL films were deposited on fluorine doped tin
oxide (FTO), glass, quartz and silicon substrates.
The self-assembled method for the preparation multilayer thin
films was as follows (Figure 1): Pt-SiPy+ Cl− and HA multilayers
deposition was manually performed upon immersing the substrate
alternately into the polyelectrolyte solutions for 4 min. After each
layer deposition, the substrate was rinsed with water at pH 2.9 and
7.9, respectively, for 10 sec and dried with air, to remove the excess of
non-adsorbed molecules. This procedure was repeated until reaching
the desired number of bilayers.
Characterization of the LbL films.—The growth of (HA/Pt-
SiPy+ Cl− )n and (Pt-SiPy+ Cl− /HA)n LbL films onto quartz substrate
was monitored via UV–Vis spectroscopy using a Varian spectropho-
tometer; model Cary 50, from 200 to 800 nm. The films with n = 30
bilayers deposited on Si substrate were also characterized by FTIR
spectroscopy in transmission mode, with spectral resolution of 4 cm−1
and 64 scans, using a Shimadzu spectrometer model Prestige 21. The
Raman scattering measurements were carried out on Stray Raman
Bruker Senterra for the (HA/Pt-SiPy+ Cl− )30 and (Pt-SiPy+ Cl− /HA)30
LbL films and drop-coated films of SiPy+ Cl− , Pt-SiPy+ Cl− and HA
(deposited on Si substrates). The morphology of the LbL films was
characterized through AFM technique using a Shimadzu Instrument
model SPM 9600 in the non-contact mode, with a Silicon SPM Sensor
(Al-coating) (NANOWORD) with thickness 4 μm, length 125 μm,
width 30 μm, frequency 320 Hz and constant force of 42.0 N m−1 .
The software used was a SPM manager, version 3.03. The average
roughness of the films was determined by this technique.
Electrochemical measurements.—All electrochemical experi-
ments, by cyclic voltammetry (CV) and differential pulse voltamme-
try (DPV), were carried out on a potentiostat/galvanostat μAutolab
(Type III Metrohm-Eco Chemie) using a software GPES version
4.9 (General Purpose Electrochemical System). A conventional
three-electrode cell was employed containing a FTO modified with
LbL or drop-coated films as working electrode (A = 0.6 cm2 ), Pt

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 Journal of The Electrochemical Society, 163 (9) B499-B506 (2016)
Figure 2. UV-Vis spectra of the influence of dipping time on the deposition
of (HA/Pt-SiPy+ Cl− )3 LbL films: 30 to 300 s.
plate as a counter electrode (A = 1.2 cm2 ) and Ag/AgCl (saturated
aqueous KCl) as a reference electrode. Differential pulse voltammo-
grams were performed in the potential range of −0.1 to 1.3 V and 0.2 to
1.5 V, respectively, using 0.1 mol L−1 electrolytes solutions (NaH2 PO4
(pH = 4.3), Na2 HPO4 (pH = 9.4), NaNO3 (pH = 6.2), KCl (pH =
6.5), and the buffers phosphate (pH = 7.5), PBS (pH = 7.2) and
Britton-Robinson (pH = 2.0) solutions, containing 62.5 μmol L−1 of
17α-ethynylestradiol (EE2), with a scan rate of 50 mV s−1 .
After the parameters optimization for the DPV (Epulse = 0.002 V,
tpulse = 0.10 s and ν = 50 mV s−1 ), the analytical curve was obtained
by addition of aliquots of different volumes of the EE2 stock solution
into the electrochemical cell (1.37 – 288 μmol L−1 ) in 0.1 mol L−1
NaH2 PO4 (pH = 7.0).
Electrochemical impedance spectroscopy (EIS) measurements
were carried out on a PGSTAT100 Autolab electrochemical sys-
tem and GPES/FRA software. For the (HA/Pt-SiPy+ Cl− )3 and (Pt-
SiPy+ Cl− /HA)3 LbL films and drop-coated films, impedance spectra
were recorded in the presence of 5.0 × 10−3 mol L−1 [Fe(CN)6 ]3−/4− ,
in 0.1 mol L−1 NaH2 PO4 (pH 7.0). EIS measurements were performed
by applying a sine wave with amplitude of 30 mV on the open-circuit
potential (OCP) in the frequency range of 10 kHz to 0.1 Hz.
Results and Discussion
Growth and spectroscopic characterization of the LbL films.—
The methodology employed in LbL self-assembly containing Pt
Figure 3. UV–Vis spectra for (a) (HA/Pt-SiPy+ Cl− )n LbL films with different number of bilayers (n = 3–42) and (b) (HA/Pt-SiPy+ Cl− )n LbL films with different
number of bilayers (n = 3–21). Insert: Relationship between the absorbance of band at ∼262 nm as a function of the number of bilayers.
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B501
nanoparticles into the SiPy+ Cl− cavities have used the H2 PtCl6 as
precursor and for the chemical reduction was formic acid.32 The char-
acterization of the hybrid Pt-SiPy+ Cl− can be found with more detail
in the literature.32 This approach has also been successfully explored
for the detection of dopamine encapsulated in liposomes38 and H2 O2
in enzymatic reactions.39
The UV-Vis spectra of the polyelectrolytes solution, Pt-SiPy+ Cl−
and humic acid (0.5 g L−1 ), data not shown, are in accordance with
the literature.32,40 The HA solution spectrum exhibits an ill-defined
band at ∼270 nm, that can be attributed to the presence of aromatic
structures.40 The spectrum of the polycation Pt-SiPy+ Cl− solution
shows an absorption band at 258 nm, which is attributed to the π−π∗
transition of the pyridine groups in SiPy+ Cl− .32
The deposition time for the construction of LbL films was opti-
mized by varying the immersion time (30 – 300 sec) for the (HA/Pt-
SiPy+ Cl− ) film containing 3 bilayers. This step was monitored by
UV-Vis technique of band at ∼262 nm, related to the π−π∗ transi-
tion of the pyridine groups in SiPy+ Cl− , as shown in Figure 2. By
increasing the dipping time, the intensity of absorbance band at ∼262
nm increases linearly up to 180 seconds. The immersion time at 210
seconds is related to the saturation of the deposited species. There-
fore, for the subsequent studies, this time was fixed at 240 seconds (4
min) in order to ensure a higher amount of immobilized species on
the substrates surface. In the literature, LbL films containing humic
acid as polyanion were assembled with dipping time of 59,11,13 or 10
minutes.41
The growth of the (HA/Pt-SiPy+ Cl− )n and (Pt-SiPy+ Cl− /HA)n
films in the optimized condition (immersion time of 4 min) was char-
acterized by UV-Vis technique, as shown in Figure 3.
The band related to the π−π∗ transition (pyridine groups of
SiPy+ Cl− ) is shifted in LbL films to higher wavelength, as compared
to solution spectrum (258 to 262 nm), which can be attributed to the
electrostatic interactions between the pyridinium ring of Pt-SiPy+ Cl−
and carboxylate anion present in the humic acid, by hydrogen bonds42
or van der Waals forces.43 As the number of bilayers increased there is
an increment in the absorbance at ∼262 nm, for the both molecular ar-
chitecture of the LbL films, as shown in the insert of Figures 3a and 3b.
The growth of the LbL films in the architecture (HA/Pt-SiPy+ Cl− )n
occurs linearly in a controlled way up to the 33 bilayers (R = 0.994).
For the (Pt-SiPy+ Cl− /HA)n LbL film the growth occurs until 9 bi-
layers (R = 0.999) and afterwards the saturation process begins. The
formation of LbL film in the architecture (HA/Pt-SiPy+ Cl− )n is more
efficient since the presence of the macromolecule (HA) on the quartz
substrate provides a greater number of anchoring sites2,5 for the Pt-
SiPy+ Cl− hybrid, ensuring a more linear and uniform growth of the
LbL films.
FTIR study was conducted to obtain details on HA-Pt-SiPy+ Cl−
interactions and in order to confirm the presence of the polyelectrolytes
 B502 Journal of The Electrochemical Society, 163 (9) B499-B506 (2016)

Figure 4. (a) FTIR spectra of KBr pellet of humic acid, drop-coated film of humic acid, (Pt-SiPy+ Cl− /HA)30 LbL film and (HA/Pt-SiPy+ Cl− )30 LbL film
in the range of 3800–800 cm−1 ; (b) FTIR spectra of drop-coated film of SiPy+ Cl− , drop-coated film of Pt-SiPy+ Cl− , (Pt-SiPy+ Cl− /HA)30 LbL film and
(HA/Pt-SiPy+ Cl− )30 LbL film in the range of 2500–550 cm−1 .

in the LbL films. Significant differences are noted in the FTIR spectra
for the drop-coated films of humic acid, SiPy+ Cl− and Pt-SiPy+ Cl− ,
and for the 30-bilayer HA/Pt-SiPy+ Cl− and Pt-SiPy+ Cl− /HA LbL
films illustrated in Figure 4.
The FTIR spectrum of neat humic acid immobilized in drop-coated
films (Figure 4a) showed a broad band at ∼3335 cm−1 , attributed to
hydrogen bond,44 which is shifted to lower wavenumber (3301 cm−1 )
in the LbL film with architecture (Pt-SiPy+ Cl− /HA)30 , and it van-
ished in the (HA/Pt-SiPy+ Cl− )30 LbL film. The bands observed at
∼2926 and 2855 cm−1 are attributed to asymmetrical and symmet-
rical stretching of the vibrations of the CH2 groups of the humic
acid,11,44 respectively. The band assigned to the vibration of aromatic
C=C of the HA44 and to the conjugated H-bonded C=O45 is ob-
served at ∼1572 cm−1 , and it is shifted to 1636 and 1701 cm−1 , in
the (Pt-SiPy+ Cl− /HA)30 and (HA/Pt-SiPy+ Cl− )30 , respectively. The
bands related to the -OH of phenols and to the COO− groups for
the HA46 (∼1453 and ∼1377 cm−1 ) are poorly defined and they
are shifted to 1487 and 1408 cm−1 for (Pt-SiPy+ Cl− /HA)30 . These
bands disappear for (HA/Pt-SiPy+ Cl− )30 film. In the HA spectrum,
the peak at 1036 cm−1 is related to the vibration of aliphatic –OH
functional group of HA,44 which is shifted to 1032 and 1072 cm−1
for the (Pt-SiPy+ Cl− /HA)30 and (HA/Pt-SiPy+ Cl− )30 architectures,
respectively. These bands are broad due to the overlap with the
Si–O–Si backbone vibrations of the SiPy+ Cl− (at 1054 cm−1 assigned
to Si–O–Si network and at 1106 cm−1 attributed to the Si–O–Si cage
structure),32,42,47 as shown in Figure 4b. The main vibrational modes
for the neat compounds and immobilized in thin films are shown in
Table I.
The changes in the spectra of the neat compounds, compared to
LbL films, may be indicative of interactions between the pyridinium
rings of Pt-SiPy+ Cl− and COO− groups of the humic acid, since the
bands attributed to these groups were the most affected.
Figure 5 show the Raman scattering spectra of drop-coated films
of Pt-SiPy+ Cl− and HA, and LbL films of (Pt-SiPy+ Cl− /HA)30 and
(HA/Pt-SiPy+ Cl− )30 deposited on silicon substrates.
The Raman spectrum of Pt-SiPy+ Cl− shows a band at ∼1028
cm−1 assigned to the breathing mode of the pyridine ring,48 which
is in agreement with the literature.32 The spectral scattering of HA
shows two main bands at 1363 and 1592 cm−1 , which are related
to sp2 sites9 and attributed to band D (ring breathing) and band G
(bond stretching either in chains or in rings),49 respectively. The
interactions between humic acid and Pt-SiPy+ Cl− are confirmed
in the Raman spectra, since the bands D and G are shifted from
1363 and 1592 to 1375 and 1589 cm−1 (Pt-SiPy+ Cl− /HA) and to
1371 and 1594 cm−1 (HA/Pt-SiPy+ Cl− ), respectively. The band at
1028 cm−1 , attributed to the breathing mode of the pyridine ring of
Pt-SiPy+ Cl− hybrid, is not observed in the spectra of LbL films.
This result indicates that the large amount of vibrational groups
present in the structure of HA may be overlapping the Pt-SiPy+ Cl−
signal.

Table I. Wavenumber and vibrational modes of the neat HA and Pt-SiPy+ Cl− and of the LbL films of (HA/ Pt-SiPy+ Cl− )30 and (Pt-
SiPy+ Cl− /HA)30 on silicon substrate.

Wavenumber (cm−1 )

Vibrational modes and groups HA Pt-SiPy+ Cl− (Pt-SiPy+ Cl− /HA)30 (HA/Pt-SiPy+ Cl− )30
H-bonded; -OH (stretching vibration)11,44 3335 — 3301 —
νas (CH2 )11,44 2926 — 2928 —
νs (CH2 )11,44 2855 — 2851 —
Pyridinium ring42 1633 1630 1710
1486
Aromatic C=C (stretching vibration)44 /Conjugated H-bonded C=O45 1572 — 1636 1701
OH of phenols and COO− groups46 1453 — 1487 —
1377 1408
Aliphatic –OH44 1036 — 1032 1072
Si–O–Si network vibration32,42,47 — 1054 1032 1072
Si–O–Si cage structure32,42,47 — 1106 1080
νs C-O 914 — 908 934
Si-O-Si (symmetric stretching)32,42,47 — 774 769 —
δ Si-H — 610 637 635

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 Journal of The Electrochemical Society, 163 (9) B499-B506 (2016) B503

sive bilayers. In the first bilayers the molecules are deposited ran-
domly until covering the whole substrate, consequently the roughness
decreases.32,42 This behavior was also observed in the studies for the
LbL films of Pt-SiPy+ Cl− with poly(vynil sulfonic acid) (PVS) or
poly(phenylene vinylene) (PPV), (PVS/Pt-SiPy+ Cl− )32 or (PPV/Pt-
SiPy+ Cl− ).39 After the first bilayers, the thickness becomes indepen-
dent of the substrate,50 occurring an increment on the roughness and
on the size of the grains with the number of bilayers, due to the
already adsorbed molecules and the non-adsorbed chains likely to ag-
gregate. In addition, the thickness of the LbL films does not depend on
the architecture, since the (HA/Pt-SiPy+ Cl− ) and (Pt-SiPy+ Cl− /HA)
containing 10 bilayers, exhibit a similar roughness, 26.601 and
25.362 nm, respectively. Therefore, the average thickness for each
bilayer is the approximately 2.6 nm. This value is similar to the
obtained for LbL film of humic acid and exfoliated Mg-Al-layered
double hydroxide nanosheets, ca. 2.1 nm.41 Crespilho et al.9 obtained
an average thickness of 0.8 nm for the bilayers of LbL film containing
humic acid and poly(allylamine hydrochloride).
Figure 5. Raman spectra for films of drop-coated Pt-SiPy+ Cl− , drop-coated
HA, (Pt-SiPy+ Cl− /HA)30 and (HA/Pt-SiPy+ Cl− )30 on silicon substrate, using
a laser of 532 nm. Electrochemical characterization of the LbL films.—The cyclic
voltammograms of (HA/Pt-SiPy+ Cl− )3 and (Pt-SiPy+ Cl− /HA)3 LbL
Table II. Root-mean-square (RMS) roughness for (Pt- films at different architectures and drop-coated films of neat humic
SiPy+ Cl− /HA)n (n = 5 and 10) and (HA/Pt-SiPy+ Cl− )n (n acid and Pt-SiPy+ Cl− were obtained in NaH2 PO4 (0.1 mol L−1 ),
= 3, 5, 10, 15 and 20) LbL films. in the range of −0.1 to 1.3 V (Figure 6), presented only capacitive
current in the potential range studied, as well as the FTO substrate.
Number of bilayers RMS roughness (nm) (5 × 5 μm) It was found in the literature that LbL films containing SiPy+ Cl− ,42
FTO 32.029 Pt-SiPy+ Cl−32 or HA41 did not present any defined redox process. The
(HA/Pt-SiPy+ Cl− )3 10.514 higher capacitive current observed to the drop-coated film of humic
(HA/Pt-SiPy+ Cl− )5 14.476 acid indicated that the presence of the HA on the surface electrode
(HA/Pt-SiPy+ Cl− )10 26.601 increases the charge density on the surface due to the negative charged
(HA/Pt-SiPy+ Cl− )15 31.588 groups present on its molecule.
(HA/Pt-SiPy+ Cl− )20 33.150 In order to understand the electrochemical properties that arise at
(Pt-SiPy+ Cl− /HA)5 14.823 molecular level in the different platforms of thin films, electrochem-
(Pt-SiPy+ Cl− /HA)10 25.362 ical impedance spectroscopy (EIS) was evaluated in the presence of
[Fe(CN)6 ]3−/4− as probe molecule. For these measurements, the fre-
Morphological characterization of the LbL films.—The morpho- quency range was chosen based on different studies with LbL films re-
logical characterization by AFM was performed in order to analyze lated in the literature.51–55 Furthermore, it is important to mention that
the effect of the architecture and number of bilayers in the roughness the electrical response depends on the frequency applied. At 1 kHz,
of the LbL films. The AFM analyses of the (Pt-SiPy+ Cl− /HA)n and the electrical response depends more strongly on the film/electrolyte
(HA/Pt-SiPy+ Cl− )n LbL films revealed a globular morphology, for interaction. However, at a low-frequency region (below 50 Hz), the
both architectures, which is similar to the reported for other LbL films electrical response is dominated by the double-layer effect.56 Thus,
in the literature.9,32,42 The morphological characterization also showed for this LbL system, it is not interesting to apply lower frequencies.
that the average roughness of the films increases with increasing num- The Nyquist diagrams (Figure 7a) show that the modification of
ber of bilayers, as listed in the Table II. FTO surface with the polyelectrolytes induces to an increase of charge
From Table II, it can be observed that the RMS roughness val- transfer resistance (Rct ) from 2.79 × 102  cm−2 (bare FTO) to 6.97 ×
ues decreased from uncovered FTO substrate (32.029 nm) to the 102 and 2.87 × 103  cm−2 for the (HA/Pt-SiPy+ Cl− )3 and (Pt-
(HA/Pt-SiPy+ Cl− )3 (10.514 nm) and then increased for the succes- SiPy+ Cl− /HA)3 , respectively. The resistance values for LbL films

Figure 6. Cyclic voltammograms of FTO, drop-coated films of Pt-SiPy+ Cl− and HA, and (HA/Pt-SiPy+ Cl− )3 , (Pt-SiPy+ Cl− /HA)3 LbL films, in NaH2 PO4
0.1 mol L−1 at pH = 7, (a) in the absence and (b) presence of 6.25 × 10−5 mol L−1 EE2 (ν = 50 mV s−1 ).

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 B504 Journal of The Electrochemical Society, 163 (9) B499-B506 (2016)
Figure 7. (a) Nyquist diagrams for the different electrodes of FTO, drop-coated film of HA and Pt-SiPy+ Cl− , (HA/Pt-SiPy+ Cl− )3 and (Pt-SiPy+ Cl− /HA)3 LbL
films in the presence of [Fe(CN)6 ]3−/4− 5.0 × 10−3 mol L−1 in 0.1 mol L−1 NaH2 PO4 , pH 7, ν = 30 mV s−1 , in the range frequency of 10 kHz to 0.1 Hz; (b)
Differential pulse voltammograms for the different electrodes of FTO, drop-coated film of Pt-SiPy+ Cl− and HA, (HA/Pt-SiPy+ Cl− )3 and (Pt-SiPy+ Cl− /HA)3
LbL films in the presence of 62.5 μmol L−1 EE2 in NaH2 PO4 , pH = 7.
comparing to bare FTO could be explained by the higher amount of
chemical species blocking the FTO surface, consequently increasing
of the Rct value. This increase is less pronounced to the architec-
ture (HA/Pt-SiPy+ Cl− )3 , since the PtNPs in the last layer assists the
charge transfer of [Fe(CN6 )]3−/4− on the surface.57 When the surface
is modified with HA in the last layer, (Pt-SiPy+ Cl− /HA)3 , there is an
increase on the Rct values because this macromolecule generates an
insulating layer on the modified surface, which should be attributed
to the repulsive electrostatic interaction caused by the anionic charge
of the film and probe molecule6,58 increasing the resistance of charge
transfer. This result can be proved by higher Rct value (4.44 × 103 )
to the HA drop-coated film. On the other hand, the Rct value to the Pt-
SiPy+ Cl− drop-coated film is 1.24 × 103 , which is lower than HA
film, evidencing the importance of the presence this hybrid to elec-
tronic transfer. However, when the (HA/Pt-SiPy+ Cl− )3 LbL film was
employed a higher decrease of the Rct value was achieved, showing
the synergistic effect for both polyelectrolytes in such architecture.
Electrochemical detection of EE2 with LbL films.—Differential
pulse voltammetry was performed using different electrode configu-
rations in presence of 62.5 μmol L−1 EE2 in 0.1 mol L−1 NaH2 PO4 .
Figure 7b displays the DPV response for (HA/Pt-SiPy+ Cl− )3 and (Pt-
SiPy+ Cl− /HA)3 LbL films, drop-coated films of HA and Pt-SiPy+ Cl− .
The DPV studies for all films were carried out in the anodic and
cathodic direction and the electrodes exhibited only an irreversible
process in the range of 0.2 to 1.5 V, corresponding to the one-electron
transfer of the OH− substituent of the EE2 ring forming the phenoxy
radical, as shown in the Equation 1.59
[1]
The electrochemical response of the bare substrate FTO in presence
of EE2 (Epa = 1.10 V and Ipa = 8.99 μA) occurred at higher positive
potential than for the drop-coated films (HA, Epa = 0.75 V and Ipa =
8.27 μA; Pt-SiPy+ Cl− , Epa = 0.92 V and Ipa = 13.63 μA). By compar-
ing with the LbL films, a lower peak potential and a higher peak cur-
rent are observed to the (HA/Pt-SiPy+ Cl− )3 and (Pt-SiPy+ Cl− /HA)3 ,
Epa = 0.74 V and Ipa = 17.21 μA and Epa = 0.81 V and Ipa = 14.08 μA,
respectively. It is suggested that the electron transfer is assisted by the
interaction of HA with Pt-SiPy+ Cl− on FTO. This can be explained
by the fact that the humic acid exhibits many functional groups, and
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consequently it can interact with Pt-SiPy+ Cl− , which is a stable and
efficient electron-transfer promoter.9 In addition to that, the PtNPs fa-
cilitates electron transfer in modified electrodes due the increasing of
the superficial area, conductivity and electrocatalytic properties.57,60
Therefore, the presence of Pt-SiPy+ Cl− in the last layer facilitated
the entrapment of hormone onto the electrode surface, thus leading to
an improved current signal,41 which is in accordance with the smaller
Rct value in presence of [Fe(CN6 )]3−/4− as observed in EIS results
(Figure 7a). The (HA/Pt-SiPy+ Cl− )3 electrode does not exhibit an
electrochemical response in the absence of EE2, even using the sen-
sitive DPV technique, then the process observed is just referred to
EE2 oxidation. The main electrochemical characteristics for the all
electrodes are shown in Table III. Subsidiary experiments performed
with the polyanion PVS instead of HA, (PVS/Pt-SiPy+ Cl− )3 (data not
shown), in the potential range between 0.6 to 1.2 V, did not present
any process, thus confirming the importance of HA presence in the
electrode.
The influence of the number of bilayers (HA/Pt-SiPy+ Cl− )n (n = 1
to 12 bilayers) for the EE2 oxidation was studied, as shown in Figure 8.
Voltammetric studies showed that the oxidation peak current for EE2
increases from 1 to 3-bilayers films, and decreases to higher bilayer
number (8 and 12). This decrease in the current intensity could be
attributed to the disorganization of the species on the electrode surface
due to the bilayers increased, which results in a higher charge transfer
resistance. Besides this, the shift of the potential values (from 0.7 to 1.1
V vs. Ag/AgCl) can be explained by the increase of the film resistance
with the number of the layers. These results are in accordance with the
AFM images (more homogeneous and less roughness, due to better
distribution of species on the surface) (Table II).
In order to optimize the electrochemical response of the (HA/Pt-
SiPy+ Cl− )3 LbL film, it was performed a study of supporting elec-
trolytes in the EE2 oxidation using DPV technique. We also examined
Table III. Anodic peak potential (Epa), anodic peak current (Ipa),
charge-transfer resistance (Rct ) and capacitance (Cdl ) for the
different electrodes of FTO, drop-coated film of Pt-SiPy+ Cl− and
HA, (HA/Pt-SiPy+ Cl− )3 and (Pt-SiPy+ Cl− /HA)3 LbL films.
Electrode Epa (V) Ipa (μA) Rct () Cdl (μF)
FTO 1.10 8.97 2.79 × 102 6.17
Pt-SiPy+ Cl− drop-coated 0.92 13.65 1.24 × 103 7.01
HA drop-coated 0.75 8.20 4.44 × 103 6.65
(Pt-SiPy+ Cl− /HA)3 0.82 14.40 2.87 × 103 5.13
(HA/Pt-SiPy+ Cl− )3 0.74 17.26 6.97 × 102 7.96
 Journal of The Electrochemical Society, 163 (9) B499-B506 (2016)
Figure 8. Differential pulse voltammograms of (HA/Pt-SiPy+ Cl− )n LbL
films with n bilayers in the presence of 62.5 μmol L−1 EE2 in NaH2 PO4 ,
pH = 7.
the ionic strength of electrolyte in DPV response. All voltammetric
measurements showed one oxidation peak in the potential range stud-
ied (data not shown). The NaH2 PO4 solution, at pH = 4.3, provided
better electrochemical response to the EE2 oxidation, due to higher
oxidation peak current (14.4 μA) and lower potential (1.06 V) in
comparison to the others electrolytes.
The influence of the pH on the oxidation peak of 62.5 μmol L−1
EE2 in NaH2 PO4 solutions of pH 1.0–10.0 is also studied (data not
shown). The increase in pH shifted the EE2 oxidation potential to
lower positive values. This is a typical behavior for the oxidation of
phenols and it was also observed by Vega et al.61 and Santos et al.25 for
the oxidation of estriol and phenolic estrogenic compounds, respec-
tively. As demonstrated, low detection potential and high peak current
was achieved using NaH2 PO4 (pH 7.0) solution as the supporting
electrolyte, which was chosen for further experiments.
Under optimized conditions, Figure 9a shows the differential
pulse voltammograms carried out at different concentrations of EE2
(1.37–21.4 μmol L−1 ) in NaH2 PO4 (pH = 7).
As observed, the DPV response depends markedly on EE2 con-
centration, without displacement of potential peak (0.74 V). The ana-
lytical curve obtained is presented in Figure 9b, with linear response
ranging from 1.37 to 21.4 μmol L−1 , with a correlation coefficient of
R = 0.998. At EE2 concentration higher than 21.4 μmol−1 , the oxida-
tion peak current is stabilized, due the low solubility of EE2 in water
Figure 9. (a) DPV responses obtained on a (HA/Pt-SiPy+ Cl− )3 LbL films at different concentrations of EE2 in a NaH2 PO4 solution (pH 7), with
Epulse = 0.002 V, tpulse = 0.10 s and ν = 50 mV; (b) Respective analytical curve.
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B505
(16.2 μmol L−1 ).62 The limits of detection (LOD) and quantification
(LOQ) were calculated according to IUPAC recommendations,63 as
LOD = 3SB /b and LOQ = 10SB /b, where SB is the standard deviation
of the blank (n = 10) and b is the slope of the calibration curve. It was
found a LOD of 1.10 μmol L−1 , LOQ of 3.68 μmol L−1 and sensitiv-
ity of 0.65 μA/μmol L−1 . These analytical responses were compared
to the glassy carbon electrodes modified with carbon nanotubes and
platinum nanoparticles, which LOD values are close to the reported
in this paper (in the range of 0.8 μmol L−1 ).23
Others modified electrodes without MNps were found in the lit-
erature for the development of electrochemical sensors for different
estrogens.23,25–27,30,64 Despite the fact that the cited studies presented
lower detection limits than the present work, some of them are based
on enzymatic reactions or require expensive and complexes platforms.
In conclusion, it can be pointed that a thin film modification of FTO
electrode with HA and Pt-SiPy+ Cl− polyelectrolytes by LbL self-
assembly method is feasible.
Conclusions
In this study, we have demonstrated the importance of
the interaction between PtNps incorporated into the 3-n-
propylpyridiniumsilsesquioxane matrix and humic acid in the elec-
trochemical oxidation of EE2. The hybrid multilayer films (HA/ Pt-
SiPy+ Cl− )n and (Pt-SiPy+ Cl− /HA)n were fabricated by the layer-by-
layer electrostatic self-assembly technique. The interaction between
these partially charged species from pyridinium rings of Pt-SiPy+ Cl−
and COO− groups of the humic acid was proved by FTIR and Ra-
man results. EIS and DPV techniques allowed the monitoring of the
successful construction of the films. The lowest Rct and the higher
current values obtained to the (HA/Pt-SiPy+ Cl− )3 LbL film in rela-
tion to (Pt-SiPy+ Cl− /HA)3 LbL film and drop-coated films containing
the polyelectrolytes separately, show the importance of the presence
of the hybrid to electronic transfer. These findings emphasized the
importance of studying the new organic-inorganic composite film by
layer-by-layer technique, which provides a uniform film growth and
a better organization of the partially charged polyelectrolytes on the
electrode surface, due to the electrostatic interactions between the
component layers. Besides, the layer-by-layer technique allows the
modular design of the architecture of the molecules on the surface.
This control enables the presence of the Pt nanoparticles in the external
layer of the (HA/Pt-SiPy+ Cl− )3 LbL film which strongly influences
the electrochemical oxidation of EE2 as demonstrated by DPV results.
In summary, the synergism between HA and Pt-SiPy+ Cl− is crucial
to improve electroactive properties of the electrode, suitable as a
promising candidate for estrogen sensing applications in pharmaceu-
tical and environmental samples.
 B506 Journal of The Electrochemical Society, 163 (9) B499-B506 (2016)
Acknowledgments
The financial support from CNPq, CAPES, CLABMU-UEPG,
INCT-INEO, Fundação Araucária and nBioNet/CAPES (Brazil) is
gratefully acknowledged.
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