Measurement 149 (2020) 106979

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Measurement
journal homepage: www.elsevier.com/locate/measurement

Determination of serotonin by using a thin film containing graphite,

nanodiamonds and gold nanoparticles anchored in casein
Matheus Mesquita Vidal Ramos a, Jefferson H.S. Carvalho a, Paulo Roberto de Oliveira b,⇑,
Bruno Campos Janegitz a
a
Department of Nature Sciences, Mathematics and Education, Federal University of São Carlos, 13600-970 Araras, SP, Brazil
b
Department of Chemistry, Federal University of Paraná (UFPR), CEP 81.531-980, Curitiba, PR, Brazil

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

Article history: The determination of serotonin has become relevant in biological fluid analysis since some neurological
Received 9 June 2019 disorders may be related to abnormal amounts of this neurotransmitter in human beings. In this work, it
Received in revised form 12 August 2019 is proposed the construction of a low-cost electrode sensitive to the quantification of serotonin by a bio-
Accepted 21 August 2019
film of graphite, nanodiamonds and gold nanoparticles anchored in casein. The casein biofilm was used to
Available online 23 August 2019
anchor the conductive nanomaterials on the GCE. Measurements were performed by differential pulse
voltammetry in which showed a linear response with LDR of 0.3 to 3.0 lmol L
1, LOD of 0.1 lmol L
1
Keywords:
and a sensitivity of 0.18 mA L mol
1. The sensor was applied for the serotonin quantification in synthetic
Biofilm
Casein
urine samples enriched with good recovery (91.4 to 103%). Therefore, the proposed electrode improved
Nanoparticles the conductivity and the electronic transfer capacity of the pristine electrode by low-cost biofilm, which
Electrochemistry could be applied in electrochemical biosensing.
Serotonin Ó 2019 Elsevier Ltd. All rights reserved.

1. Introduction alternative due to simplicity in their preparation by using rela-

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter
synthesized in central and peripheral nervous systems, appetite,
sleep, and mood regulator, and is related to various neuropsychi-
atric disorders such as depression, anxiety, and eating disorders
[1–3]. Once the excessive production of 5-HT can promote the car-
cinoid tumors, it is necessary the determination of this analyte in
biological fluids. Diagnosis can be made by detecting 5-HT levels
in the urine, giving relevant information about the clinical condi-
tion of patients [4]. In this context, some analytical techniques
are employed in the determination of serotonin in biological fluids,
such as luminescence [5], chromatography-electrochemical detec-
tion [6], fluorescence [7], capillary electrophoresis [8] and electro-
chemistry [9]. Each technique has advantages and disadvantages.
The use of electrochemical techniques, such as voltammetric in
environmental, biological and pharmaceutical analysis, is increas-
ingly earning notoriety [10–12]. This is due to the advantages that
these techniques have over others, such as good selectivity, high
sensitivity, minimum sample preparation steps, and mainly, low-
cost [11]. Thus, the use of electrochemical sensors is an attractive
⇑ Corresponding author.
E-mail address: proliveira.ufpr@gmail.com (P.R. de Oliveira).
tively low-cost materials [13,14].
Concerning the electrochemical sensors, different strategies are
employed in the proposition of the electrodes for the detection of
several analytes of interest, including 5-HT [2,15–18]. In this
way, a multifaceted approach is the use of conductive thin films
within nanomaterials [19,20], which exploit the electrical and syn-
ergistic properties of its components [20,21]. An example is a
nanocrystalline diamond or nanodiamond (ND), a novel carbon
material that has several advantages over the same content in bulk
scales, such as large surface area, chemical stability, and low toxi-
city [22]. Also, due to the small size, the nanoparticles have a large
number of carbon atoms with sp2 hybridization and p bonds on
their surface, ensuring to the ND an excellent electrical conductiv-
ity [23–25], wide potential range and low background current in
aqueous solutions.
Gold nanoparticles is another material that has been widely
used in science due to their electronic, optical, and biocompatibil-
ity properties [14,26]. They have been utilized in many areas, such
as physics, chemistry, biology, medicine, and materials science
[27]. Besides, the reduced size of these particles promote an excel-
lent performance to electrochemical measurements in the deter-
mination of electroactive species that have been verified in the
literature. For example, Caetano et al. [28] used a carbon glassy
modified electrode with gold nanoparticles supported on carbon

https://doi.org/10.1016/j.measurement.2019.106979
0263-2241/Ó 2019 Elsevier Ltd. All rights reserved.
2 M.M.V. Ramos et al. / Measurement 149 (2020) 106979

nanotubes in the determination of dopamine. The presence of the
nanoparticles increased the surface area of the base electrode 14
times, which resulted in a significant improvement in the perfor-
mance due to the increase in the active surface area of the elec-
trode. Similarly, Winiarski et al. [29] developed an electrode with
AuNP stabilized in silsesquioxane. This electrode was applied to
the voltammetric determination of sulfite ions by electrochemical
reduction. The presence of the nanoparticulate material promoted
higher sensitivity and electrocatalytic properties to this electrode,
verified an overpotential of
0.20 V when compared to the
unmodified CPE. The success of the application of nanomaterials
is dependent not only on their inherent properties but also of their
efficient immobilization and dispersion in the composite material
[30]. The use of biofilms in the composite materials synthesis has
gained significant importance in the preparation of the sensor. This
is due to the possibility to use organic and inorganic species to
improve the selectivity and/or electrochemical performance of
the electrochemical sensors and biosensors [31–33].
An exciting example of biopolymer that has been used for bio-
film formation is casein (CSN), a phosphoprotein present in mam-
malian milk, representing 80% of the total amount of protein that
composes it [34]. The CSN, like other milk proteins, has been used
for food supplementation to gain muscle mass [35]. Thus synthesis
of CSN thin film is possible in which can be incorporated to con-
ductive materials. Although it is an insulating material, the use of
CSN films can be successfully employed in the immobilization
and stabilization nanoparticles without decrease of the properties
of the immobilized materials [36,37]. In this work, we propose the
synthesis of the modification of glassy carbon electrode (GCE) with
a conductive thin nanoparticulate film based on the incorporate of
nanodiamonds, gold nanoparticles, and graphite anchored in CSN.
GCE features good electrical conductivity, low-cost compared to
other electrodes such as gold or platinum, and easy surface
renewal by simple polishing [38,39]. Also, it has low roughness,
and thereby its real surface area may be similar to the geometric
area. The modification aims at improving the increased surface
area of the pristine electrode by promoting excellent performance
in the determination of 5-HT in urine samples.
2. Experimental procedure
2.1. Chemicals
stirring for 90 min. The CSN/ethanol dispersion was cooled in an
ice bath to 25.00 °C. Posteriorly, 20.00 mL this dispersion was
added in 60.00 mL of ultrapure water at 50 °C under constant stir-
ring and subsequently cooled to room temperature. The NDs-
AuNPs-Gr-CSN dispersion was prepared by simply mixing of
1.00 mg of Gr, 1.00 mg of NDs, 500 lL of the AuNPs dispersion,
and 1.00 mL of the CSN dispersion. The NDs-AuNPs-Gr-CSN disper-
sion was placed in an ultrasonic bath followed by vortex for 90 min
to the homogenization. The NDs-AuNPs-Gr-CSN/GCE were
obtained by simply casting of 5 lL of NDs-AuNPs-Gr-CSN (lowest
amount added with good accuracy) on the GCE surface, and it
was allowed to dry for 6 h at room temperature. The steps are
shown in Fig. 1.
2.4. Morphological characterization
The Scanning Electron Microscopy (SEM) and dark-field scan-
ning of the electrodes were performed in the FEI Scanning Electron
Microscope, model Inspect S 50 with a FEG (field emission gun)
electron source (Tokyo, Japan) with the resolution of 1.0 mm and
2 mm.
2.5. Voltammetric measurements
The voltammetric measurements were performed by a poten-
tiostat/galvanostat Autolab PGSTAT204 (Eco Chemie) Metrohm
coupled to a notebook and controlled by Nova 2.0.1. Software. A
set of three electrodes in a 10.00 mL electrochemical cell were
used. A glassy carbon electrode (Ø = 3 mm) modified with the film
NDs-AuNPs-Gr-CSN was used as working electrode, Ag|AgCl|
3.0 mol L
1 KCl was used as the reference electrode, and platinum
was used as counter electrode (Pt).
2.5.1. Voltammetric characterization
The cyclic voltammetric measurements (CV) was performed in a
potential range that was possible to observe the faradaic oxidation
process of the 5-HT. Thus, the potential range was conducted from
0.15 V to 0.60 V under a scan rate of 50 mV s
1. The differential
pulse voltammetry (DPV) technique was used for the 5-HT quan-
tification. The voltammetric measurements were performed, rang-
ing from 0.25 V to 0.55 V under-evaluated conditions of pulse

All chemicals used are of analytical grade. Chloroauric acid

tetrahydrate (HAuCl4), nanodiamonds, graphite, potassium chlo-
ride (KCl), Serotonin, Casein, Sodium phosphate (Na3PO4), potas-
sium ferricyanide (K3[Fe(CN)6]), potassium ferrocyanide (K4[Fe
(CN)6]) were supplied by Sigma-Aldrich (San Luis, Missouri, EUA)
and Fluka (São Paulo, São Paulo, Brazil). The solutions were pre-
pared by using deionized (DI) water (18 MO cm, Millipore).

2.2. AuNPs synthesis

The AuNPs synthesis was performed using the method

described by Turkevich [40]. The procedure consists of the addition
of 4.00 mL of an aqueous solution of 0.05 mol L
1 HAuCl4 in 200 mL
of ultrapure water at 85.00 °C under constant stirring. After, it was
added 2.00 mL of 0.30 mol L
1 sodium citrate kept under stirring.
After 4 min, the reaction medium was placed in an ice bath for
cooling to room temperature (25.00 °C).

2.3. NDs-AuNPs-Gr-CSN/GCE preparation

Fig. 1. Illustration of the steps of obtaining NDs-AuNPs-Gr-CSN/GCE and 5-HT
voltammetric determination. (a) The mixing process of the components and
The CSN dispersion was prepared by adding 0.06 g casein in sonication. (b) GCE modification with NDs-AuNPs-Gr-CSN film by drop-casting and
60.00 mL ethanol 70% (v/v) and heated at 76.00 °C under constant subsequent drying at room temperature. (c) 5-HT voltammetric determination.
 M.M.V. Ramos et al. / Measurement 149 (2020) 106979
amplitude, pulse time, and scan rate. The pulse amplitude values
were studied from 10 to 100 mV, the pulse time from 10 to
100 ms, and the scan rate was from 5 to 25 mV s
1. The influence
of the pH on the voltammetric response to the NDs-AuNPs-Gr-
CSN/GCE was also investigated. For this, DPV measurements were
obtained in 0.10 mol L
1 phosphate buffer in the presence of
1.0 mmol L
1 5-HT changing the pH solution at pH 5.1, 6.1, 6.9,
7.5, and 7.9.
2.5.2. Analytical performance of the NDs-AuNPs-Gr-CSN/GCE
The analytical curve was performed by DPV measurements in
the presence of different concentrations of 5-HT (0.3 mmol L
1,
0.74 mmol L
1, 1.0 mmol L
1, 1.5 mmol L
1, 2.0 mmol L
1, and
3.0 mmol L
1) employing the best measurement conditions
obtained in the characterization studies. The 5-HT determination
was performed by DPV in synthetic urine solution at four concen-
tration levels (1.0 mmol L
1, 1.5 mmol L
1, 2.0 mmol L
1, and
3.0 mmol L
1) in the same analytical curve conditions.
The synthetic urine solution for 5-HT recovery test was pre-
pared in ultrapure water using CaCl22H2O, NaCl, Na2SO4, NaHPO4,
KCl, NH4Cl, urea and creatinine were adjusted to pH 6.00, as indi-
cated by Laube et al. [41].
3. Results and discussion
3.1. Electrochemical performance of NDs-AuNPs-Gr-CSN/GCE by 5-HT
determination
This manuscript aims at the application of a low-cost and
straightforward electrode modified with biocomposite for the
determination of 5-HT. This conception is of high relevance within
the current scientific context that search the determination of spe-
cies of biological interest employing sensitive electrodes with the
use of nanostructured materials, such as thin films modified with
nanoparticles or conducting nanostructures [42–44]. To make this
proposal viable, studies were carried out to evaluate the ability of
the modified electrode to determine 5-HT, as well as its character-
ization. As cited in the introduction section, 5-HT is an essential
neurotransmitter that can be found in biological fluids. In this con-
text, electrochemical measurements were performed to evaluate
the potentiality to 5-HT detection in 0.1 mol L
1 KCl, and the pres-
ence of 1.0 mmol L
1 5-HT and the voltammograms are shown in
Fig. 2. First, as comparison, cyclic voltammetric measurements
were made by using the following electrodes: NDs-AuNPs-Gr-
CSN/GCE; ND-AuNPs-CSN/GCE; Gr-CSN/GCE and pristine GCE.
Measurements of cyclic voltammetry in the presence of 5-HT
(Fig. 2A) show the presence of a faradaic process only for the
CGE electrodes and the NDs-AuNPs-Gr-CSN/GCE. The observed sig-
Fig. 2. (A) Cyclic voltammograms and (B) Differential pulse voltammograms; for (a) NDs-AuNPs-Gr-CSN/GCE; (b) GCE; (c) NDs-AuNPs-CSN/GCE and (d) Gr-CSN/GCE in the
presence of 1.0 mmol L
1 5-HT and 0.10 mol L
1 KCl Conditions: CV – scan rate = 50 mV s
1; DPV – scan rate = 10 mV s
1, pulse amplitude = 25 mV, modulation time = 50 ms.
3
nal is more pronounced for the NDs-AuNPs-Gr-CSN/GCE when
compared to GCE, at the same concentration of 5-HT. This signal
refers to the 5-HT electrochemical oxidation since the electrodes
evaluated did not present a faradaic signal (not shown) in this
region of potential in the conditions employed in the absence of
5-HT (blank). The increase in the signal can be explained by the
increased surface area and synergic effect presented by the modi-
fied electrode when in the presence of nanoparticles and graphite,
as well as by the greater surface area promoted by the presence of
AuNPs. The technique used for quantification was the DPV because
it showed to be more sensitive. The pulsed technique presents the
response current with less contribution to the capacitive current.
Also, the 5-HT oxidation reaction is irreversible when compared
with square wave voltammetry (SWV). The oxidation reaction
shown above in Scheme 1 represents the electronic transfer of
two electrons between the 5-HT molecule and the working elec-
trode, generating a faradaic (peak) process at 0.40 V as observed
in Fig. 2B. The highest peak intensity is presented by the NDs-
AuNPs-Gr-CSN/GCE, followed by the unmodified electrode (GCE),
the NDs-AuNPs-CSN/GCE and the Gr-CSN/GCE. The best perfor-
mance to the NDs-AuNPs-Gr-CSN/GCE can be explained by the
presence of gold and diamond nanoparticles that increase the
active surface area of the electrode. This increase of the current
in the presence of the nanoparticles is related to the mass transport
process [45].
On the other hand, the Gr-CSN/GCE presented the lowest cur-
rent intensity that decreased the current intensity in comparison
to the base electrode. This result can be explained by the formation
of the CSN film that probably decreased the conductivity of the
GCE and influences the electronic transfer of the 5-HT oxidation
reaction. The addition of nanoparticles to CSN/GCE promoted the
electrochemical improvement of this electrode. This can be
observed for the NDs-AuNPs-CSN/GCE, proving the improvement
of modified electrodes with conductive nanoparticulate materials.
The occurrence of a 5-HT oxidation reaction in the mentioned
potential is in agreement with other papers of the literature that
report this reaction [18,46]. The reaction is exemplified in
Scheme 1, in which occurs the 5-HT oxidation reaction by deple-
tion of 2 electrons and 2 protons [47].
3.2. Voltammetric characterization
The best electrochemical response of the NDs-AuNPs-Gr-CSN/
GCE in comparison to the GCE was attributed to better electron
transfer, such as the larger surface area promoted by the presence
of the nanoparticles. Thus, to prove such assumptions, the electro-
chemical characterization of these electrodes in the presence of
probes was also performed. The electrochemical characterization
4 M.M.V. Ramos et al. / Measurement 149 (2020) 106979
Scheme 1. Mechanism of 5-HT oxidation in 5-HT quinoneimine.
of NDs-AuNPs-Gr-CSN/GCE and GCE was carried out by cyclic
voltammetry in the presence of a solution of K3[Fe(CN)6] and
K4[Fe(CN)6] both 1.0 mmol L
1 in 0.10 mol L
1 KCl solution chang-
ing the scan rates. In the voltammograms shown in Fig. 3, it is pos-
sible to verify the increase of the anodic and cathodic peak current
to the NDs-AuNPs-Gr-CSN/GCE electrode compared to the unmod-
ified electrode (GCE). This increase is due to the high electroactive
area after modification with NDs and AuNPs. The presence of the
nanaoparticles provided a better and more effective rate of elec-
tron transfer [48], as already observed for the measurements in
the presence of 5-HT.
The electrode characterization was performed by CV at different
scan rates (Fig. SM1). The slope values (ip vs m1/2) allowed the esti-
mate of the electroactive area for the NDs-AuNPs-Gr-CSN/GCE and
GCE electrode from the Randles-Sevcik equation (Eq. (1)).
ip ¼ 2:69  10  n 5 3=2
AD 1=2
 C0  m 1=2
ð1Þ
2
Where ip is the peak current (A), A is the electroactive area (cm ), C
is the probe concentration used, D is the diffusion coefficient
(7.6 mcm2 s
1), n is the number of electrons and v is the scan rate
(V s
1). The estimated areas were 0.10246 cm2 and 0.07331 cm2
for NDs-AuNPs-Gr-CSN/GCE and GCE, respectively, representing
an increase of 28%. The observed behavior was linear, indicating
that the mass transfer occurred only by diffusion [49]. The results
have indicated a synergic effect of NDs and AuNPs present in the
proposed modified electrode, such as observed to NDs-AuNPs-Gr-
CSN/GCE voltammetric behavior in parallel to the GCE unmodified
behavior (Fig. 3).
3.3. Morphological characterization
The morphology of the NDs-AuNPs-Gr-CSN/GCE was evaluated
by scanning electron microscopy (SEM) analysis. Samples of the
30
NDs-AuNPs-Gr-CSN/GCE
20 GCE
10
i / µA
0
-10
-20
-30
-0.4 -0.2 0.0 0.2 0.4 0.6 0.8
-1
E / V (vs Ag|AgCl|3.0mol L KCl)
Fig. 3. Cyclic voltammograms for NDs-AuNPs-Gr-CSN/GCE (black) and GCE (red)
electrodes in the presence of equimolar solution of 1.0 mmol L
1 [Fe(CN)6]3
/4
and
0.10 mol L
1 of KCl and m = 50 mV s
1. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
CSN dispersion, the NDs-AuNPs-CSN film, and the NDs-AuNPs-
Gr-CSN film were submitted to this analysis. Fig. 4 shows the
SEM images.
In Figs. 4(a) and 2(b) (casein dispersion) is possible to observe
the presence of a rugous surface from casein clusters in film forma-
tion. Figs. 4(c) and 2(d) (NDs-AuNPs-CSN dispersion) show the
aggregates of NDs and heterogeneous structures formed by NDs
and AuNPs. In Fig. 4(e) it is possible to observe a higher concentra-
tion of carbon material in NDs-AuNPs-Gr-CSN than in Fig. 4(c) (due
to the addition of Gr). By comparing Fig. 4(f), the NDs-AuNPs-Gr-
CSN film presents heterogeneous structures formed by the four
materials, being that the carbon materials are more exposed due
to their higher concentration in comparison to the AuNPs. Besides,
darkfield scanning measurements (Fig. 4d and f) showed the pres-
ence and crystallinity of the gold nanoparticles (bright particles). It
is evident the presence of the nanoparticles at the edge of the CSN
film, which makes them more exposed to the species in solution.
The SEM results showed the presence of dispersed nanomaterials
on the film structure formed by CSN and Gr. This proves the effec-
tiveness of the composite film to immobilize or anchor the conduc-
tive species, responsible for the significant improvement of the
modified electrode.
3.4. Optimization of experimental parameters
The evaluation of the best experimental condition for the deter-
mination of 5-HT was carried out by studies of experimental
parameters used in chemical and electrochemical measurements
by DPV. Thus, the influence of the pH values of the measurement
medium, the scan rate (v), pulse amplitude and pulse modulation
time on the analytical signal and resolution of the voltammograms
(Fig. SM2).
The study of the effect of the pH of the solution was performed
voltammetric analyses in the presence of 0.10 mol L
1 phosphate
buffer and 1.0 mmol L
1 5-HT, in different pH: 5.7, 6.1, 6.9, 7.4
and 7.9, as observed in Fig. 5A and B.
In Fig. 5A, it is possible to observe the influence of the pH value
on the voltammetric measurements regarding the peak intensity
and the faradaic peak potential values. In this study, it can be seen
that the anodic peak intensity decreases as the pH increases
(Fig. 5B). The oxidation potential of 5-HT is observed the decrease
of this value with increasing pH, to stabilization at pH 7.4, probably
by less amount of protonated 5-HT in this condition (pKa  10)
[50]. This behavior is verified in other papers [46] that attribute
this to the proton participation in the oxidation reaction of 5-HT,
as shown in Scheme 1. In this case, the measurements carried
out at pH 5.7 and 6.1 showed a higher current intensity, in which
the best measurement condition was attributed to pH 6.1 since it
presents a lower oxidation potential (0.38 V vs. Ag|AgCl|3.0 mol L
1
KCl) compared to pH 5.7 (0.42 V vs. Ag|AgCl|3.0 mol L
1 KCl). This
pH value coincides with the value of the synthetic urine used
(pH 6.0). The oxidation process of 5-HT that occurs near 0.40 V is
in agreement with other works that use the same reference elec-
trode [18,46].
It was also observed the influence of the parameters of the DPV
on the voltammetric response to 5-HT oxidation, such as anodic
peak intensity and oxidation potential (data not shown). The eval-
uated ranges of each parameter, as well as the condition that pre-
sented the best responses are summarized in Table 1.
3.5. Analytical performance of the NDs-AuNPs-Gr-CSN/GCE and urine
analysis
The analytical curve was performed by DPV using the optimized
conditions in the presence of different concentrations of 5-HT in
the range of 0.3 mmol L
1 to 3.0 mmol L
1. The obtained
 M.M.V. Ramos et al. / Measurement 149 (2020) 106979 5

Fig. 4. Electron microscopy images of (a) and (b) CSN dispersion at 2 lm and 500 nm, respectively; (c) bright-field scanning of NDs-AuNPs-CSN at 2 lm; (d) dark-field
scanning of NDs-AuNPs-CSN at 2 lm; (e) bright-field scanning of NDs-AuNPs-Gr-CSN at 1 lm and (f) dark-field scanning of NDs-AuNPs-Gr-CSN at 1 lm.

0.5
A B
0.4

0.3
i / µA

0.2

0.1

0.0
5.5 6.0 6.5 7.0 7.5 8.0
pH
Fig. 5. A) DP voltammograms and B) Correlaction plot of peak oxidation intensity vs pH for NDs-AuNPs-Gr-CSN/GCE in 0.10 mol L
1 phosphate buffer and in the presence of
1.0 mmol L
1 5-HT at pH 5.7, 6.1, 6.9, 7.4 and 7.9. Conditions: modulation time = 10 ms, pulse amplitude = 80 mV and v = 5 mV s
1.

voltammograms (n = 3) and the analytical correlation curve are
shown in Fig. 6A and B, respectively.
The NDs-AuNPs-Gr-CSN/GCE showed linearity (R2 = 0.998) of
the peak intensity vs. 5-HT concentration in a linear dynamic range
(LDR) from 0.3 to 3.0 mmol L
1 with a sensitivity of 0.18 mA L mol
1.
From the analytical curve, equation it was obtained a limit of quan-
tification (LOD) and limit of quantification (LOQ) of 0.1 mmol L
1
and 0.33 mmol L
1, respectively [51]. The LOD and LOQ were esti-
mated from three and ten times the standard deviation of the
blank, respectively, divided by the analytical curve sensitivity
6 M.M.V. Ramos et al. / Measurement 149 (2020) 106979

Table 1 extrapolated in the regression equation obtained by the analytical

DPV experimental parameters for the detection of 5-HT with the electrode NDs- curve, and the recovery percentage value was obtained from the
AuNPs-Gr-CSN/GCE.
calculated and added 5-HT concentration. The values obtained
Parameters Evaluated Range Selected can be seen in Table 3.
Pulse amplitude (mV) 10–100 80 In healthy humans, the serotonin is quickly metabolized by the
Modulation time (ms) 10–100 10 liver resulting in a very low urinary amount of 5-HT in the non-
v (mV s
1) 5–25 5 metabolised form [58,59]. However, in doping or physiological dis-
pH 5.7–7.9 6.1
orders cases, the concentration can increase to 32 lmol L
1 [60],
that allow the identification of these problems by monitoring of
5-HT in dilute urine samples. The recovery values ranged from
91.4% to 103%. Therefore, we concluded that the ample matrix
[51]. The merit parameters obtained for the determination of 5-HT did not significantly interfere in the voltammetric signals. So, we
by NDs-AuNPs-Gr-CSN/GCE are better or comparable with other did not choose to perform the studies of possible interferents of
papers in the literature that perform the electrochemical determi- urine concomitant species in the presence of 5-HT. However, this
nation of 5-HT (Table 2). It is essential to highlight the proposed does not exclude the possibility of interference from other pharma-
electrode is easy to prepare and presents low cost when compared ceutical compounds and metabolites. This study will maybe
to other electrodes for the same purpose. Besides, comparing to perform in other papers after this one.
other determination techniques of 5-HT (Table 2), such as UV spec-
trophotometry, ultra-performance liquid chromatography-mass
spectrometry (UPLC-MS/MS), and spectrofluorimetric, the pro-
Table 3
posed method presents similar or inferior performance regarding Recovery values in the detection of 5-HT by sensor NDs-AuNPs-Gr-CSN/GCE in
LOD, for example. However, the proposed method presents advan- synthetic urine samples.
tages inherent to voltammetric techniques, such as low cost, ana-
Sample Added (lmol L
1) Recovery (lmol L
1) %Recovery
lytical frequency and operational simplicity [52].
A 1.00 0.91 91.4
The application of the NDs-AuNPs-Gr-CSN/GCE sensor was per-
B 1.50 1.47 98.4
formed in synthetic urine samples (pH 6.1) enriched with 5-HT at C 2.00 2.06 103
four levels concentration: 1.0 mmol L
1, 1.5 mmol L
1, 2.0 mmol L
1 D 3.00 3.02 100
and 3.0 mmol L
1 (A–D) (n = 3). The peak current intensities were

Table 2
Comparison of the LOD of the proposed method with other previously published papers.

Determination Electrode/Detector Electrochemical Technique LOD Ref

(mmol L
1)
Electrochemical F-MWCNTs/BR9 DPV 9.00 [53]
Spectrophotometric UV–Vis – 2.30 [54]
Electrochemical GN-SPEs CV 1.00 [55]
Spectrometric UPLC-MS/MS – 0.013 [56]
Electrochemical GCE/MWCNT-NiO SWV 0.120 [46]
Spectrofluorimetric Spectrofluorometer – 0.002 [57]
Electrochemical NDs-AuNPs-Gr-CSN/GCE DPV 0.100 *

F-MWCNTs/BR9 – basic red 9 film (BR9)/functionalized multiwall carbon nanotubes; UV–Vis – Ultraviolet–Visible; GN-SPEs – Modified screen-printed electrodes based on
oxidized graphene nanoribbons; UPLC-MS/MS – ultra-performance liquid chromatography-mass spectrometry; GCE/MWCNT-NiO – nickel oxide nanoparticles/MWCNT on
glassy carbon electrode; CV – Cyclic voltammetry; SWV – Square wave voltammetry.

Fig. 6. A) DP voltamograms and B) Analytical correlation curve for detection of different 5-HT concentrations employing the NDs-AuNPs-Gr-CSN/GCE (n = 3). Conditions:
0.10 mol L
1 phosphate buffer (pH 6.1), modulation time = 10 ms, pulse amplitude = 80 mV and v = 5 mV s
1.
 M.M.V. Ramos et al. / Measurement 149 (2020) 106979
4. Conclusion
The construction of the proposed electrode occurred, low cost
and with reasonable accuracy, in which were obtained films of
the composite material with a high distribution of the nanoparti-
cles, which promoted a larger surface area of the electrode. The
results evidenced the potential of CSN in the CSN in the formation
of stable biofilms capable of of immobilizing other materials. The
presence of the nanoparticulate content promoted to the base elec-
trode great capacity of electron transfer of the oxidation reaction of
5-HT. The proposed methodology proved to be useful in the quan-
tification of 5-HT in synthetic urine samples successfully. Thus, the
proposed sensor presented high electronic transfer capacity and
excellent performance in the determination of 5-HT which is
shown with a good alternative for the determination of 5-HT and
the methodology may be useful in 5-HT detecting as disease mar-
ker even in the complex urine matrix.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper
Acknowledgments
The authors are grateful to CAPES (finance code 001), FAPESP
(2017/21898-6, 2017/21097-3 and 2019/01844-4) and CNPq for
financial support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.measurement.2019.106979.
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