Journal of Pharmaceutical and Biomedical Analysis 196 (2021) 113912

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Journal of Pharmaceutical and Biomedical Analysis

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

Determination of paracetamol using a sensor based on green

synthesis of silver nanoparticles in plant extract
Felipe Zamarchi, Iolanda Cruz Vieira ∗
Department of Chemistry, Federal University of Santa Catarina, 88040-900, Florianópolis, SC, Brazil

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

Article history: The biosynthesis of nanometals using a plant extract is simple, efficient, fast, cost-effective and eco-
Received 30 July 2020 friendly. In this study, a pine nut extract (Araucaria angustifolia) was obtained and used as a reducing and
Received in revised form 15 January 2021 stabilizing agent in the synthesis of silver nanoparticles. An electrochemical sensor based on the silver
Accepted 18 January 2021
nanoparticles obtained and exfoliated graphite nanoplatelets applied to a glassy carbon electrode was
Available online 21 January 2021
developed for the determination of paracetamol. To optimize the synthesis of the silver nanoparticles,
important factors such as temperature, extract:water ratio, silver nitrate concentration and extract sta-
Keywords:
bility time were studied. The factors influencing the performance of the sensor were studied in detail and
Paracetamol
Green synthesis the results demonstrated good repeatability and electrode-to-electrode repeatability (relative standard
Nanoparticles deviations of 1.8 and 4.0 %, respectively). Under optimized conditions, there was a linear response to
Electrochemical sensor paracetamol concentrations of 4.98 × 10−6 to 3.38 × 10-5 mol L-1 , with a detection limit of 8.50 × 10-8
mol L-1 . No reports on the biosynthesis of AgNPs using Araucaria angustifolia could be found in the lit-
erature. The sensor developed showed good stability and was used successfully for the quantification of
paracetamol in pharmaceutical products.
© 2021 Elsevier B.V. All rights reserved.

1. Introduction activated carbon have also been reported as an effective sorbent

In recent years, considerable attention has been directed toward
the application of nanostructured materials (scale of 1−1000 nm).
This is because they have catalytic, electronic, magnetic and opti-
cal properties that are strongly dependent on their small size and
chemical composition. These properties give the materials a high
active surface area, catalytic activity and good electrical conduc-
tivity. Metal nanoparticles have applications in diverse areas, such
as cosmetics, coatings, biotechnology, packaging, electronics, drug
delivery systems and biosensors [1–6].
Nanomaterials have attracted attention due to their excel-
lent properties and have been explored as promising materials
in the magnetic dispersive micro-solid phase extraction method
coupled with high performance liquid chromatography. In this con-
text, Asfaram et al. [7–10] proposed a magnetic Cu:CuO-graphene
oxide nanocomposite for the separation and pre-concentration of
polycyclic aromatic hydrocarbons [7]. The CuO/ZnO@Fe3 O4 -carbon
nanotubes and Fe3 O4 @SiO2 @Ti-MOF were used as an efficient sor-
bent for the pre-concentration and determination of chlorogenic
acid and caffeic acid in medical extract of plants and in food and
water samples [8,9]. Manganese-oxide nanoparticles loaded on
∗ Corresponding author.
E-mail address: iolanda.vieira@ufsc.br (I.C. Vieira).
combined with ultrasound for the extraction and determination of
a trace amount of thymol and carvacrol in methanolic extracts of
Thymus daenensis, Salvia officinalis, Stachys pilifera, Satureja khuzis-
tanica, mentha and water samples [10].
Silver nanoparticles (AgNPs) have been extensively studied
for many decades due to their unique features and wide range
of applications. They have antibacterial, anticancer and antimy-
cotic activity and are used in catalysis, imaging and biosensors.
In the literature, several methods for the synthesis of AgNPs
have been reported, including chemical, electrochemical, photo-
chemical and sonochemical approaches and procedures involving
laser irradiation, microwave irradiation and thermal decomposi-
tion, among others. Chemical reduction is the most common and
general method where the AgNPs are prepared in two steps: reduc-
tion (e.g., using borohydride, hydrazine, sodium citrate, citric acid
or oxalic acid) and stabilization (e.g., with poly(vinyl alcohol),
poly(ethylene glycol), surfactants, ionic liquids, dendrimers and
polymers) [11–16].
The use of plant extracts in the synthesis of nanometals has been
widely explored by researchers from different areas of knowledge
and this alternative has led to procedures for obtaining metallic
nanoparticles that offer simplicity, speed and non-toxicity [17–25].
The first plant material employed in the biosynthesis of silver
nanoparticles was alfalfa sprouts [25] and later different plant parts,
such as leaves, fruits, flowers, seeds, stems and roots, were used

https://doi.org/10.1016/j.jpba.2021.113912
0731-7085/© 2021 Elsevier B.V. All rights reserved.
F. Zamarchi and I.C. Vieira
to synthesize this metal. Examples include leaf extracts of Pelargo-
nium graveolens, Carica papaya, Curcuma longa, Eucalyptus hybrid,
Moringa oleífera, Melia azedarach, Piper longum, Polyalthia longifo-
lia, Cinnamomum camphora, Eucalyptus citriodora, seed extracts of
Anacardium occidentale L, Syzygium cumini (L.), root extracts of Oci-
mum sanctum and flower extracts of Nelumbo nucifera (lotus) and
Calotropis procera [15–23,26–33]. It is known that plant extracts can
act as reducing and stabilizing agents in the synthesis of nanoparti-
cles. They contain phenolic acid, flavonoids, enzymes, amino acids,
proteins, alkaloids, terpenoids, alcoholic compounds and polysac-
charides, which are mainly responsible for the reduction of ionic
into bulk metallic nanoparticles. In addition, chlorophyll and quinol
contribute to the reduction of silver ions and stabilization of the
nanoparticles [13,15–19].
The biosynthesis of AgNPs using plant extracts is simple, effi-
cient, fast, cost-effective and eco-friendly. Additionally, there is
great interest in the use of metallic nanoparticles in the construc-
tion of sensors. The development of electrochemical sensors is an
efficient alternative for the determination in several types of sam-
ples [34–44]. Some advantages are quick response, good sensitivity
and low-cost equipment and reagents, and there is generally no
need for sample pre-treatment.
The use nanostructured materials in the construction of
electrochemical sensors has resulted in major advances in
terms of stability and sensitivity. In the literature, differ-
ent platforms have been reported, such as carbon paste
electrodes modified with a nanohybrid composite: platinum
nanoparticle/polyoxometalate/two-dimensional hexagonal boron
nitride nanosheets for the determination of N-hydroxysuccinimide
[36], copper oxide-decorated reduced graphene for the simulta-
neous determination of cholesterol, ascorbic acid and uric acid
[37], incorporation of an NiO-doped Pt nanostructure hybrid
for the determination of cysteamine [38] and nanostructured
orthorhombic vanadium pentoxide for the detection of hydro-
gen peroxide [39]. Electrochemical aptamer-based methods for
the determination of bisphenol A were constructed using gold
nanoparticles immobilized on a conjugate of multiwalled car-
bon nanotubes and thiol-functionalized magnetic nanoparticles
[40], and gold nanoparticles immobilized on functionalized cop-
per magnetic nanoparticles and multiwall carbon nanotubes [41].
An electrochemical sensor based on multiwall carbon nanotubes
and magnetic nanoparticles functionalized with tannic acid and Au
nanoparticles deposited on a glassy carbon electrode was develop-
ment in order to determine small amounts of 17␣ – ethinylestradiol
[42], a platform containing a nanocomposite of graphene oxide/zinc
based on a metal-organic framework prepared through a simple
solvothermal method was employed for the monitoring of As(III)
in environmental water samples [43], and a nanocomposite based
on multi-walled carbon nanotube/thiol functionalized magnetic
nanoparticles as an immobilization platform and gold nanoparti-
cles was used for the detection of bisphenol A [44].
In this study, an extract of pine nut (Araucaria angustifolia)
was obtained and used as a reducing and stabilizing agent in the
synthesis of AgNPs. To optimize the synthesis, important factors
such as temperature, extract:water ratio, AgNO3 concentration and
extract stability time were studied. The AgNPs were then used to
build an electrochemical sensor for the detection of paracetamol
(N-acetyl-p-aminophenol), a drug with analgesic and antipyretic
properties widely used to relieve headaches and reduce fever
[45–47]. To improve the performance of the proposed sensor, exfo-
liated graphite nanoplatelets (xGnPs) were added. This material is
composed of sp2 hybridization carbons and consists of layers of
stacked graphene that partially preserve their electrical, thermal
and mechanical properties. Due to their conductive characteristic,
increased surface area and low cost, the use of xGnPs has become
established as an alternative in the development of new sensors for
2
Journal of Pharmaceutical and Biomedical Analysis 196 (2021) 113912
application in electrochemistry [48,49]. No reports on the biosyn-
thesis of AgNPs using Araucaria angustifolia and their use in the
development of a sensor (AgNP-xGnP/GCE) could be found in the
literature. The sensor was used successfully for the quantification
of paracetamol in commercial pharmaceutical products.
2. Experimental
2.1. Reagents, solutions and samples
All reagents were of analytical grade and used without further
purification. All aqueous solutions were prepared with ultrapure
water, obtained from a Milli-Q system (Millipore, USA), with
a resistivity of 18.2 M cm−1 . Silver nitrate and paracetamol
(PA) were obtained from Sigma-Aldrich. The exfoliated graphite
nanoplatelets (xGnPs) were obtained from XG Science. Acetate
buffer solutions (pH 5.0 and 4.0) and phosphate buffer saline - PBS
(pH 7.0, 7.4 and 8.0) were prepared at a concentration of 0.1 mol
L−1 and were used for optimization of the plant extract-stabilized
AgNPs and as supporting electrolytes in the optimization of the
method. Three Brazilian medicines containing paracetamol (A, B
and C) were acquired from a local drugstore in Florianópolis (Santa
Catarina, Brazil) and analyzed using the proposed AgNP-xGnP/GCE
sensor and an analysis method obtained from Pharmacopoeia. Pine
nuts (Araucaria angustifolia) were purchased from a local producer
in Florianópolis, Brazil, from which an extract was obtained and
used as an AgNP reducing and stabilizing agent.
2.2. Obtention of plant extract and biosynthesis of AgNPs
Initially, the pine nuts were washed several times with ultra-
pure water and finely cut into small pieces. In this next step, 6 g of
the Araucaria angustifolia pieces was macerated and 5.0 ml of ultra-
pure water was added. The extract was filtered and centrifuged
at 18,000 rpm for 3 min at 25 ◦ C. The resulting extract was used
as a stabilizing and reducing agent in the synthesis of the silver
nanoparticles.
The biosynthesis was carried out according to the following pro-
cedure: AgNPs were formed spontaneously by the addition of 5.0 ml
of an aqueous solution of 5.0 mmol L−1 silver nitrate to 1.0 ml of
the extract (diluted by a factor of 10) under mechanical stirring
at 45 ◦ C for 35 min. The synthesis was considered complete after
acquiring a brown-yellow colored solution indicating the forma-
tion of silver nanoparticles (AgNPs) and this was verified by UV–vis
spectroscopy.
2.3. Instrumentation
Spectrophotometric analysis of the AgNPs was performed on
a Cary 60 UV–vis spectrometer (Agilent Technologies, USA) with
a quartz cell (optical path of 1.0 cm). The morphology and par-
ticle sizes of the nanomaterial samples were characterized using
transmission electron microscopy (TEM) and the analysis was per-
formed with a JEOL JEM-1011 transmission electron microscope,
at an acceleration voltage of 100 keV, in the Central Laboratory
of Electron Microscopy at the Federal University of Santa Cata-
rina (Florianópolis, Brazil). The samples were deposited onto a
carbon-coated copper grid. The grids were placed over filter paper
to remove the excess material and air dried.
Square-wave voltammetry (SWV), differential pulse voltam-
metry (DPV) and cyclic voltammetry (CV) were performed
with an AUTOLAB101 potentiostat/galvanostat (Eco Chemie, The
Netherlands) and electrochemical impedance spectroscopy (EIS)
was performed with an AUTOLAB128 N potentiostat/galvanostat
(Eco Chemie, The Netherlands), both equipped with the data
processing software NOVA (version 1.10). The experiments were
F. Zamarchi and I.C. Vieira Journal of Pharmaceutical and Biomedical Analysis 196 (2021) 113912

carried out using a three-electrode system with the proposed
AgNP-xGnP/GCE sensor, assembled using a glassy carbon electrode
(GCE, diameter 2.0 ± 0.1 mm) as the working electrode, Ag/AgCl
(3.0 mol L−1 KCl) as the reference electrode and platinum foil as
the counter electrode.
2.4. Construction of the AgNP-xGnP/GCE sensor and
electrochemical measurements
3. Results and discussion
3.1. Optimization of the biosynthesis of AgNPs stabilized with
plant extract
A wide variety of plant species are found in nature and many
are candidates for the synthesis of AgNPs. In this study, a pine
nut (Araucaria angustifolia) extract was selected for this purpose.

Initially, the glassy carbon electrode (GCE) was prepared for

the construction of the AgNP/GCE and AgNP-xGnP/GCE sensors by
mechanically polishing the surface with an aqueous suspension of
alumina (0.05 mm) for 2 min using a polishing cloth, followed by
rinsing with ultrapure water and ethanol. The GCE was then soni-
cated in an ultrasonic bath, in ultrapure water, for 2 min to remove
residual particles.
In an Eppendorf, 2.0 mg of the exfoliated graphite nanoplatelets
(xGnPs) was mixed with 100.0 ␮L of AgNP solution (diluted in ultra-
pure water in a 1:1 ratio). This material was then stirred in a Vortex
for 2 min and in a sonicator for 5 min. The sensor was prepared by
dropping 3.0 ␮L of this nanomaterial onto the GCE surface and the
electrode was then left in a desiccator for 10 min. The electrochem-
ical sensor (AgNP-xGnP/GCE) was then ready for use.
The electrochemical measurements (CV, SWV, DPV and EIS)
were taken at room temperature (≈ 25 ◦ C) in an electrochem-
ical cell containing 10 ml of the supporting electrolyte solution
(PBS 0.01 mol L−1 , pH 7.5) and the required volume of analyte
(standard solution or paracetamol samples). A stirring time of 60 s
was applied to homogenize the solutions and the peak currents
obtained for paracetamol on the proposed sensor surface were used
for the determination of this analyte. The CV measurements were
recorded by cycling the potential between 0.0 and +0.800 V at a
scan rate of 100 mVs−1 . The SWV analysis was carried out applying a
scanning potential of +0.100 to +0.550 V, frequency of 10 Hz, ampli-
tude of 100 mV and increment of 1 mV, after successive additions
of the analyte. EIS measurements were used for the characteriza-
tion of the sensor and were performed in a 0.1 mol L−1 KCl solution
containing 5.0 mmol L−1 [Fe(CN)6 ]3−/4− , in open circuit mode, with
a frequency range of 0.1–100,000 Hz and amplitude of 10 mV.
Several tests were carried out in order to validate the pro-
posed method. The repeatability (6 successive measurements using
the same sensor) and reproducibility (4 sensors prepared inde-
pendently) were evaluated based on the results for the relative
standard deviation (RSD). The linear range of the calibration curve
was evaluated using the correlation coefficient (r2 ), which enabled
the determination of the limits of detection and quantification. The
paracetamol content of the pharmaceutical samples obtained using
the sensor was compared with the value obtained using a method
given in the Pharmacopeia.

2.5. Preparation of pharmaceutical sample and determination of

paracetamol

The content of one tablet (samples A, B and C) was ground with

a mortar and pestle and the powder obtained was dissolved in
100 ml of ultrapure water. This solution was sonicated for 5 min
in an ultrasonic bath and filtered through filter paper. Aliquots
(5.0 mL) of these pharmaceutical samples were transferred to a
100 ml volumetric flask that was filled with ultrapure water. In the
next step, 100 ␮L was transferred to the cell containing the sup-
porting electrolyte and quantified after successive additions of a
standard solution of paracetamol. After each addition, SWV read-
ings were recorded from +0.100 to +0.800 V. A spectrophotometric Fig. 1. Influence of (A) plant extract:water (mL) ratio on the absorbance of the AgNPs
method for paracetamol determination available in the Pharma- in the solutions, (B) concentration of AgNO3 (mol L−1 ) solutions on the stability of the
AgNPs over time. Insert: absorbance of the solutions on the first day; (C) synthesis
copeia [50] was used to compare the analytical results with those o
temperature ( C) on the stability of the AgNPs over time. Insert: influence of the
obtained using the proposed sensor. temperature on the absorbance on the first day.

3
F. Zamarchi and I.C. Vieira
Fig. 2. (A) TEM micrograph of the plant extract-stabilized AgNP, (B) particle size histogram based on approximately 300 particles, (C) and (D) TEM micrographs of the
AgNP-xGnP.
In addition to the nature of the plant extract, in each part of the
plant there are different types and concentrations of biomolecules,
according to the need for plant tissue and the type of stress to which
the plant has been subjected [11–21]. Therefore, to obtain a good
performance in the biosynthesis of the AgNPs, parameters such as
extract:water ratio, extract stability time (days) according to con-
centration of AgNO3 (mol L−1 ) and extract stability time (days)
according to synthesis temperature (◦ C) were evaluated.
The molecular absorption spectrum of the AgNPs synthesized
in Araucaria angustifolia was obtained 30 min after the synthesis at
a wavelength of 415 nm. Spectrophotometric measurements were
used to monitor the formation and stability of the nanoparticles.
Fig. 1(A) shows the variation in the absorbance according to the
extract:water ratio (1:3, 1:1, 3:1 and 1: 0), using 6.0 mmol L−1 sil-
ver nitrate solution and a temperature of 45 ◦ C. As can be seen, the
highest absorbance was obtained using the 1:0 extract:water ratio.
The effect of the concentration of the AgNO3 solution (0.05, 0.25,
0.5, 2.5 and 5.0 mmol L−1 ) on the formation of AgNPs was then eval-
uated using an extract:water ratio of 1:0 and temperature of 45 ◦ C.
On the first day of the synthesis, after 45 min under slight agitation
(best time for formation of AgNPs in this study), only the 0.25 and
0.50 mmol L−1 solutions of silver nitrate were found to lead to the
formation of AgNPs. A study on the stability of the AgNPs obtained
in these two solutions was conducted for a 40-day period. As can
be seen in Fig. 1(B), the absorbance signal increased for up to 12
and 19 days for the synthesis of AgNPs with 0.25 and 0.50 mmol
4
Journal of Pharmaceutical and Biomedical Analysis 196 (2021) 113912
L−1 silver nitrate solutions, respectively, and after that time the
absorbance remained constant (0.25 mmol L−1 ) and a there was a
slight increase for nanoparticles synthesized with a 0.50 mmol L−1
solution. The best response and stability were obtained using a sil-
ver nitrate solution of 0.50 mmol L−1 (see inset in Fig. 1B). Therefore,
this was the concentration of silver nitrate used for the biosynthe-
sis of the nanoparticles used in the subsequent experiments in this
study.
The biosynthesis of AgNPs at different temperatures (25, 35, 45
and 55 ◦ C) was studied using an extract:water ratio of 1:0 and the
0.5 mmol L−1 silver nitrate solution with a reaction time of 45 min
and constant agitation. As shown in Fig. 1C, over a period of 36
days these solutions were analyzed at a wavelength of 415 nm. It
can be observed that the absorbance measurement for the solu-
tions of AgNPs initially shows a considerable increase and then
remains constant after day 10. This indicates that temperature is
not a crucial factor in the synthesis process, since an increase in
absorbance occurred in the solutions kept at room temperature.
Thus, in the subsequent experiments, a temperature of 45 ◦ C was
selected.
It has been reported in the literature that parameters such as
the concentration of metal ions, reaction period and extract com-
position have a strong impact on the size, shape and morphology of
AgNPs. Several researchers have applied temperatures of 20–100 ◦ C
for the synthesis of AgNPs and the incubation times ranged from
15 min to 24 h [9,15].
F. Zamarchi and I.C. Vieira
Fig. 3. (A) Schematic representation of the reaction involving paracetamol on the surface of the sensor; (B) cyclic voltammogram at 100 mV s−1 , (C) SWV using the (a) bare
GCE, (b) AgNP/GCE, (c) AgNP-xGnP/GCE and (D) voltammograms obtained applying the electroanalytical techniques: (a) SWV and (b) DPV in PBS solution (0.1 mol L−1 , pH
7.4) containing 1.48 × 10-5 mol L−1 paracetamol.
3.2. Morphological characterization of AgNPs and AgNP-xGnP
Fig. 2 shows the TEM micrographs obtained for the AgNPs syn-
thesized with the Araucaria angustifolia extract and the AgNP-xGnP.
As seen in Fig. 2A, the AgNPs have an almost spherical shape, vary-
ing in size and well dispersed without the formation of aggregates.
The size distribution of the nanoparticles obtained is shown in
the histogram in Fig. 2B. The AgNPs showed an average diame-
ter of approximately 91.0 ± 0.5 nm, which was estimated from a
set of 300 particles present in arbitrarily chosen areas of the TEM
images, with the size distribution being reasonably represented by
a Gaussian curve. On the other hand, the xGnPs in the nanoma-
terial (AgNP-xGnP) was observed in the TEM images as rectangles
composed of multiple layers of graphene, varying from tens to hun-
dreds of nanometers. This typical lamellar structure of graphite
nanoplates can be seen in Figs. 3C and 3D (100 nm and 1 ␮m), along
with the presence of AgNPs attached to the graphene sheets.
3.3. Sensor functioning and voltammetric determination of
paracetamol
The working principle of AgNP-xGnP/GCE is illustrated in
Fig. 3(A) and is based on the process of oxidation-reduction of N-
acetyl-p-aminophenol. In this process, this substance is oxidized
to N-acetyl-p-benzoquinoneimine on the surface of the proposed
5
Journal of Pharmaceutical and Biomedical Analysis 196 (2021) 113912
sensor and this product is electrochemically reduced to N-acetyl-
p-aminophenol at a potential of +0.40 V vs. Ag/AgCl. The cyclic
voltammogram (Fig. 3B) was obtained for a 9.09 × 10−5 mol L-1
solution of PA solution in phosphate buffer saline (0.1 mol L-1 , pH
7.4) at a scanning speed of 100 mV s-1 . The presence of AgNPs and
xGnPs used in the modification of the GCE, improved the properties
(larger surface area and higher conductivity), facilitating the elec-
tron transfer reaction and this resulted in increased stability and
sensitivity.
To study the contribution of each modifier (AgNPs and xGnPs),
different electrodes were prepared and their performance was
compared to the electrode without modification (GCE, electrode
“a”). Thus, the electrodes investigated were AgNP/GCE, AgNP-
xGnP/GCE, xGnP/GCE and GCE. Electrochemical measurements
were taken by SWV, in triplicate, in the potential range of +0.200
to +0.500 V vs. Ag/AgCl, also using a 9.09 × 10−5 mol L-1 solution
of paracetamol in phosphate buffer saline (0.1 mol L-1 ; pH 7.4).
Fig. 3(C) shows that the use of AgNPs as a modifier (AgNP/GCE, sen-
sor “b”) allowed an increase in the current exceeding that obtained
with the GCE, which can be attributed to the intrinsic characteristics
of the NPs, such as high conductivity and catalytic capacity. Due to
the high conductivity and the properties of the xGnPs, electrode “c”
(AgNP-xGnP/GCE) presented a current gain and thus the response
was much higher than those obtained with the other electrodes,
being about 40 times greater than that of the electrode without
F. Zamarchi and I.C. Vieira Journal of Pharmaceutical and Biomedical Analysis 196 (2021) 113912

Fig. 4. Results for effect of (A) xGnP (0.5 – 2.0 mg), (B) support electrolyte (PBS solution and acetate) and pH (5.0 to 8.0) using 9.09 × 10−5 mol L-1 paracetamol solution,
analyzes performed in triplicate. Inset shows voltammograms obtained using the proposed sensor.

modification. A xGnP/GCE was also constructed. However, the film

on the electrode surface did not show stability and leaching of
the material was observed during electrochemical measurements.
The addition of AgNPs (AgNP-xGnP/GCE) favored the stability and
enhanced the analytical response. Based on the results obtained,
the present study confirms the advantages of using these nanoma-
terials in the development of the proposed sensor.
Fig. 3(D) shows the voltammograms obtained for the electrode
modified with AgNP-xGnP/GCE using SWV and DPV, indicating that
the response obtained for the SWV was superior to that obtained
using DPV. Therefore, SWV was selected as the technique to be used
in the subsequent experiments.
The effects of the SWV parameters, such as frequency
(10−100 Hz), amplitude (1−10 mV) and increment (1−10 mV),
were investigated using the proposed electrode (AgNP-xGnP/GCE)
in PBS solution (0.1 mol L−1 , pH 7.4) containing 1.48 × 10-5 mol L−1
paracetamol. For all measurements, the responses obtained were
evaluated in terms of resulting current, peak profile and noise in Fig. 5. Electrochemical impedance spectra for the (a) bare GCE, (b) AgNP/GCE and (c)
the voltammograms. The best voltammetric profile was obtained AgNP-xGnP/GCE in 0.1 mol L−1 KCl solution containing 5.0 mmol L−1 [Fe(CN)6 ]3−/4− ,
in open circuit mode, with a frequency range of 0.1 to 100,000 Hz and amplitude of
with a frequency of 10 Hz, amplitude of 100 mV and increment of 10 mV.
1 mV, and thus these values were applied in the subsequent exper-
iments.
3.5. Electrochemical characterization of the sensor

EIS measurements were performed to assess the charge transfer

3.4. Study of xGnPs, pH and supporting electrolyte
In this study, exfoliated graphite nanoplatelets (xGnPs) were
used in the construction of the sensor, together with the AgNPs, in
order to obtain better performance and a lower detection limit. As
previously reported, xGnPs are obtained by exfoliating the graphite,
providing a large surface area. Thus, the effect of the amount of
xGnPs, which varied from 0.25 to 2.0 (mg), on the response of the
proposed AgNP-xGnP/GCE was studied. The analytical response for
9.09 × 10−5 mol L-1 paracetamol in PBS solution (0.1 mol L-1 , pH 7.4)
increased significantly with 2.0 mg xGnPs, as shown in Fig. 4(A). The
construction of the sensor with a larger quantity was not possible
due to the inhomogeneity of the solution. Thus, 2.0 mg was the
quantity selected for the construction of the AgNP-xGnP/GCE.
The appropriate choice of the supporting electrolyte and the pH
of the solution is very important to ensure good sensitivity of the
electrochemical method. The effect of the supporting electrolyte
and pH on the analytical response for PA was investigated by SWV,
using 0.1 mol L−1 PBS solutions (pH 7.0, 7.4 and 8.0) and 0.1 mol L−1
acetate buffer (pH 5.0 and 6.0). In Fig. 4(B), it can be observed that
the highest current response for PA was obtained with the 0.1 mol
L−1 phosphate buffer saline (pH 7.4) and this electrolyte and pH
were thus applied in the subsequent experiments.
resistance (Rct) for each construction step of the proposed sen-
sor. Fig. 5 shows the Nyquist diagrams (-Z vs. Z ) obtained for: (a)
unmodified GCE, (b) AgNP/GCE and (c) AgNP-xGnP/GCE in KCl solu-
tion (0.1 mol L−1 ) containing Fe(CN)6 3-/4- (5.0 mmol L−1 ). For the
modified electrodes, the electrochemical impedance spectra, using
Fe(CN)6 3-/4- as a redox species, show that the Rct values obtained
were: unmodified GCE (4.525 k), AgNP/GCE (Rct ≈ 0.445 k)
and (c) AgNP-xGnP/GCE (Rct ≈ 0.051 k). As can be seen from the
Nyquist diagram, after the addition of AgNPs there was a reduction
in the electrical resistance when compared to the GCE without any
modification and this is due to the conductive capacity of AgNPs.
However, with the addition of the graphite nanoplatelets, faster
electron transfer and, consequently, lower electrical resistance was
observed for the proposed sensor (AgNP-xGnP/GCE). This indicates
that the combined addition of AgNPs and xGnPs as modifiers facil-
itates the process of electronic charge transfer.
3.6. Calibration curve, reproducibility studies and application to
commercial drug
Under the selected conditions mentioned above, the square-
wave voltammograms for paracetamol in phosphate buffer saline
(0.1 mol L−1 , pH 7.4) were obtained employing the proposed AgNP-

6
F. Zamarchi and I.C. Vieira
Fig. 6. (A) Square wave voltammograms obtained using the AgNP-xGnP/GCE in PBS solution (0.1 mol L−1 , pH 7.4) containing different concentrations of paracetamol (a)
4.98 × 10-6 , (b) 7.44 × 10-6 , (c) 9.90 × 10-6 , (d) 1.48 × 10-5 , (e) 1.96 × 10-5 , (f) 2.44 × 10-5 , (g) 2.91 × 10-5 and (h) 3.38 × 10-5 mol L-1 at frequency 10 Hz, pulse amplitude 100 mV
and scan increment 1 mV and (B) corresponding calibration curve.
Table 1
Determination of paracetamol (mg) in commercial pharmaceutical products using
the proposed sensor and Pharmacopeia method [50].
Paracetamol (mg)
Sample Label Pharmacopeia Sensora
A 500 514 ± 3.3 493 ± 9.1
B 750 746 ± 0.7 730 ± 12.6
C 500 509 ± 1.3 447 ± 11.1
a
Mean standard deviation; n = 3.
b
Relative error (%): Re1 =sensor label value; Re2 =sensor pharmacopeia.
xGnP/GCE in the potential range of +0.100 to +0.550 V vs. Ag/AgCl.
The experiments were carried out in triplicate. The voltammograms
obtained are shown in Fig. 6A and the corresponding calibra-
tion curve for paracetamol (Fig. 6B) was linear for concentrations
of 4.98 × 10-6 to 3.38 × 10-5 (i = -1.647 × 10-5 (±0.001) + 5.695
(±0.05) [PA], r2 = 0.9980; where i is the resultant peak current
in ␮A and [PA] is the paracetamol concentration in mol L−1 ). The
detection limit (three times the blank signal/slope) was calculated
using the linear range and the value obtained was 8.50 × 10-8 mol
L−1 .
A repeatability study was carried out through 6 successive mea-
surements by SWV, using the same sensor in phosphate buffer
saline (0.1 mol L−1 ; pH 7.4) containing 1.48 × 10-5 mol L−1 of parac-
etamol. An RSD of 1.8 % was obtained in this study, verifying good
accuracy of the measurements. The reproducibility between the
sensors was also investigated considering 4 sensors prepared inde-
pendently. Good reproducibility was obtained, with an RSD value
of 4.0 % for the sensors analyzed.
In order to evaluate the efficiency of the proposed sensor, the
quantification of paracetamol in three commercial medicine sam-
ples (A, B and C) was performed with the AgNP-xGnP/GCE and the
results were compared with those obtained with the Pharmacopeia
method [37] used as a comparison. The results obtained were also
compared with the concentration given on the product label and
are shown in Table 1. As can be seen, the paracetamol content
determined by the sensor was consistent with that specified by the
manufacturer and with the value determined by the comparative
method. These results indicate that the methodology is suitable for
the determination of paracetamol in drugs.
4. Conclusions
In this study, we developed a novel sensor based on the mod-
ification of the surface of a glassy carbon electrode (GCE) with a
nanostructured film of silver nanoparticles that were synthesized
in a plant (Araucaria angustifolia) extract and exfoliated graphite
nanoplatelets were added. This sensor presented attractive ana-
Journal of Pharmaceutical and Biomedical Analysis 196 (2021) 113912
lytical parameters including good repeatability and reproducibility
(1.8 % and 4.0 %, respectively), linear response to a relatively wide
range of the paracetamol concentrations (4.98 × 10−6 to 3.38 × 10-5
mol L-1 ), low detection limit (8.50 × 10-8 mol L-1 ) and good preci-
Re1 and Re2 (%)b sion and accuracy. In addition, the sensor was successfully applied
for the determination of paracetamol in commercial pharmaceuti-
−1.4 and -4.1
−2.6 and -2.1 cal products. This good performance is mainly due to the presence
−10.6 and -12.2 of the nanomaterials used in the modification of the GCE, which
improved the properties (e.g., larger surface area and enhanced
conductivity), facilitating the electron transfer reaction. Other
notable advantages of the method are the ease of sensor construc-
tion and the low cost of instrumentation and reagents. Therefore,
the sensor developed in this study can be considered a convenient
tool for the fast and accurate determination of paracetamol in phar-
maceutical samples.
Author statement
The authors declare that the manuscript entitled “Determina-
tion of paracetamol using a sensor based on green synthesis of
silver nanoparticles in plant extract” is original and has not been
submitted for review in other journals. The authors also declare
that all of them contributed to the design and implementation of
the research, to the analysis of the results and to the writing of the
manuscript.
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 for financial support received from
CNPq (Process 442609/2014-0 and 304120/2017-0) and also schol-
arships granted by CNPq to FZ. This research was also supported by
the Central Laboratory of Electron Microscopy, Federal University
of Santa Catarina State (Florianópolis, SC, Brazil).
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