Food Chemistry 437 (2024) 137698

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Electrochemical determination of Roxarsone using preconcentration-based

signal amplification on modified screen-printed electrode
Luís M.C. Ferreira a, b, Paulo R. Martins c, Cristiane G. Silva c, Luiz H. Marcolino-Junior b,
Marcio F. Bergamini b, Fernando C. Vicentini a, *
a
Center of Nature Sciences, Federal University of São Carlos, Rod. Lauri Simões de Barros km 12, 18290-000 Buri, SP, Brazil
b
Laboratory of Electrochemical Sensors (LabSensE) - Department of Chemistry, Federal University of Paraná, 81.531-980, Curitiba, PR, Brazil
c
Institute of Chemistry, Federal University of Goiás, Av. Esperança, Goiania, GO 74690-900, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: A lab-made screen-printed electrode based on poly(ethylene terephthalate) (PET) substrate modified with a
Roxarsone hybrid film containing gold nanoparticles-decorated graphene (AuNPs-GRA/PET-SPE) was employed for the
Screen-printed electrode voltammetric determination of Roxarsone (ROX) in chicken purge and river water samples. The electrode
Graphene
exhibited an increased electroactive area and enhanced charge transfer due to the nanostructured matrix. The
Gold nanoparticles
electrochemical determination involved a preconcentration approach with a reduction step of ROX at a constant
Preconcentration
potential of − 0.6 V, followed by voltammetric sweep towards the oxidation of the adsorbed hydroxylamine at
0.32 V. The methodology achieved a limit of detection of 60 nM and 97 nM for ROX in diluted river water and
chicken purge samples, respectively. This effective methodology offers a promising tool for monitoring ROX
levels in environmental and food samples.

1. Introduction highly sensitive detection (e.g., fluorescence and inductively-coupled

plasma spectroscopy) are commonly employed for ROX determination
Roxarsone (ROX) is an organoarsenic drug which has been exten­ (Conklin, Shockey, Kubachka, Howard, & Carson, 2012; Liu, Yu, Song,
sively used in the poultry industry to treat coccidiosis caused by enteric Qiu, Sun, Li, et al., 2008; Roerdink & Aldstadt, 2004; Rosal, Momplaisir,
parasites alongside with other anticoccidial drugs (Schmidt, 2013). Its & Heithmar, 2005). However, these techniques are typically associated
use was also associated to other beneficial economic features, such as to large equipment, being restricted to laboratorial environment.
improved growth rates in broiler chickens (Li, Wang, Li, & Wu, 2012). On the other hand, electrochemical approaches have been explored
However, due the concern about potential release and migration of aimed their potential for in loco analysis, especially due the advantage of
inorganic arsenic in food products and the environment, the production associated to miniaturization and portability without loss of sensitivity
of ROX have been discontinued and banned in several countries as (Kokulnathan, Rajagopal, Wang, Huang, & Ahmed, 2021; Tyszczuk-
Australia, Canada, China, EU, Malaysia and US. Nevertheless, in other Rotko & Gorylewski, 2022; Waris, Baig, Sirajuddin, Kazi, Solangi, Sid­
countries including Argentina, Brazil and Mexico, ROX is still found in diqui, et al., 2016). Furthermore, electrode performance can be
use (Hu, Cheng, Tao, & Schnoor, 2019; R. Tang, Prommer, Yuan, Wang, enhanced via surface modification, particularly by incorporating
Sun, Jamieson, et al., 2021). Although ROX concentrations in poultry various nanoarchitetures to achieve the required sensitivity for quanti­
meat are considered low, they are not negligible, and there is no fication of ROX at environmental levels, including lanthanum molyb­
consensus about the effects of long exposure to human health (Konkel, date nanoroses (Vinoth, Govindasamy, Wang, Alothman, & Alshgari,
2016), being imperative the monitoring of ROX levels in food products 2021), tungsten disulfide (M. Govindasamy, Wang, Jothiramalingam,
and farms in order to prevent human and environment contamination. Noora Ibrahim, & Al-Lohedan, 2019), rare earth vanadate nanoparticles
For this reason, sensitive analytical methods for ROX determination (Balasubramanian Sriram, Baby, Hsu, Wang, & George, 2023; B. Sriram,
have gained attention. Separation-based techniques, such as capillary Kogularasu, Hsu, Wang, & Sheu, 2022), and hybrid frameworks that
electrophoresis, liquid, ion, and gas chromatography associated to combine a carbon material matrix with inorganic nanostructures (Chen,

* Corresponding author.
E-mail address: [email protected] (F.C. Vicentini).

https://doi.org/10.1016/j.foodchem.2023.137698
Received 25 July 2023; Received in revised form 8 September 2023; Accepted 6 October 2023
Available online 9 October 2023
0308-8146/© 2023 Elsevier Ltd. All rights reserved.
L.M.C. Ferreira et al. Food Chemistry 437 (2024) 137698

Rajaji, Chen, Chinnapaiyan, & Ramalingam, 2019; Ferreira, Silva, electron microscopy JEOL model JEM-2100EX-II and scanning electron
Augusto, Gomes-Junior, Farra, Silva, et al., 2022; Mani Govindasamy, microscopy JEOL, model JSM 6360-LV, respectively. The acquired im­
Rajaji, Wang, Chang, Ramalingam, & Chan, 2020; Nikhil, Srivastava, ages were analyzed using ImageJ 1.53 k software (developed by W.S.
Srivastava, Srivastava, & Prakash, 2022; Rajaji, Ganesh, Kim, Govin­ Rasband from the U.S. National Institutes of Health in Bethesda, Mary­
dasamy, Alshgari, & Liu, 2022; Tamilalagan, Akilarasan, Chen, Govin­ land, USA). X-ray diffraction (XRD) was performed with a Bruker D8
dasamy, Lin, Alzahrani, et al., 2023). Discover diffractometer with copper Cu-Kα1 radiation.
Regarding hybrid electrode films, metallic nanoparticles, especially
gold nanoparticles (AuNPs) arranged in a decorated framework in gra­ 2.3. Fabrication and modification of PET-SPE electrode
phene (and graphene derivates) have been widely employed in elec­
troanalysis. The selection for this type of architecture is based on its The fabrication of PET-SPE was conducted according to the pro­
numerous advantages usually assigned to synergistic effects that cedure previously reported (Ferreira, Reis, Martins, Marcolino-Junior,
enhance the overall electrode performance when compared to individ­ Bergamini, Camargo, et al., 2023). The conductive carbon ink was
ual nanomaterials, which includes higher surface area, faster electron prepared by mixing 700 mg of graphite powder and 300 mg of nail
transfer rates, catalytic properties toward high sensitivity, and enhanced polish, resulting resulted mixture with 70% (m/m) carbon content.
stability, since the graphene usually works as a passivating/dispersing Then, 800 µL of ethyl acetate was added to reduce viscosity, and the
medium for metallic nanoparticles, preventing aggregation (Fritea, mixture was manually homogenized. Polyethylene terephthalate (PET)
Bănică, Costea, Moldovan, Iovan, & Cavalu, 2018; Miao, Feng, Jin, He, substrate was prepared by cutting the soft-drink bottles in a 25 × 35 mm
Liu, Du, et al., 2015; Santos, Wong, Ferreira, Soares, Fatibello-Filho, pieces, which were washed and sanded. An adhesive vinylic paper was
Moraes, et al., 2021; Xia, Guo, Zhao, Xu, Yu, Xu, et al., 2014). More­ finely cut to use as electrode mask using a cutting printer (Silhouette,
over, some properties of AuNP-graphene composite can be modulated in Cameo 3). After that, the graphite ink was applied on the prepared
order to increase adsorption of molecules, leading to material accumu­ substrate using a spatula. After removing and discarding the mask, a
lation on the electrode surface and can be used as analytical feature device consisting of a three-electrode array with a centered working
(Govindhan & Chen, 2015; Huang, Liu, Li, Zhang, & Yao, 2015; Kang­ electrode (Ø = 5.0 mm) was obtained. Silver ink was used to cover the
kamano, Numnuam, Limbut, Kanatharana, & Thavarungkul, 2017; Tan, pseudo-reference electrode using a fine tip brush.
Hu, Wu, Li, Li, Yu, et al., 2015; Yola, Eren, & Atar, 2015). For modification of PET-SPE, a colloidal solution of AuNPs was
Here, we present the electrochemical determination of ROX using a prepared using a modified borohydride reduction procedure (Ferreira,
lab-made screen-printed electrode (SPE) made by serigraphy of a et al., 2023): 590 μL of the 0.05 mol/L HAuCl4 solution was added to 90
conductive carbon ink on poly(ethylene terephthalate) substrate (PET- mL of water while undergoing continuous vigorous stirring at room
SPE), which was modified with hybrid film of gold nanoparticles- temperature, resulting in a pale-yellow colored solution. After 1 min of
decorated graphene (AuNPs-GRA) to enhance the ROX current. A stirring, 1.0 mL of 38.8 mmol/L sodium citrate was added into the
comprehensive redox mechanism for ROX was stablished using cyclic mixture, which was stirred for an additional minute. Finally, 1.0 mL of
voltammetry. The analytical procedure for ROX determination was 0.075% sodium borohydride in 38.8 mmol/L sodium citrate was added
based on the multiple process associated to the analyte, starting with the to the mixture, leading to an instantaneous change in dispersion color to
reduction of nitro group under the constant potential, thus setting an ruby red. The mixture was then stirred for more 10 min. A volume of 1.0
electrochemical preconcentration step, followed by anodic square-wave mL of AuNPs solution was mixed to 1.0 mg of graphene and sonicated for
voltammetric sweep toward oxidation of the adsorbed material. After 30 min at room temperature to produce the modifying dispersion. Then,
optimization of the electrochemical parameters the procedure was 10 µL of this dispersion was cast onto the working electrode surface,
applied in the analysis of ROX in real samples. which was left to dry at room temperature, producing the AuNPs-GRA/
PET-SPE.
2. Experimental
2.4. Samples preparation
2.1. Reagents and solutions
The chicken breast meat purge was extracted after thawing the
Gold(III) chloride (HAuCl4), sodium borohydride, hydrated sodium sample at room temperature from the spontaneous secretion of the
citrate, potassium ferricyanide, potassium ferrocyanide, potassium pinkish protein liquid. An aliquot of 100 μL of this liquid was diluted in
chloride, and Roxarsone (ROX) (pharmaceutical secondary standard) 10 mL of 0.1 M HCl. The spiked sample was prepared by adding 5 μM of
were obtained from Sigma-Aldrich. Graphene powder (grade AO-3, 12 ROX to a fraction of the diluted sample. The river water sample was
nm sheet thickness) was purchased from Graphene Supermarket®. diluted in a 1:10 ratio in 0.1 M HCl, and the corresponding spiked
Graphite powder was acquired from Fischer Chemical (USA), and silver sample was prepared by adding 5 μM of ROX to the undiluted sample.
ink from EMS (USA). Colorless nail polish for nail care (Risqué™, São
Paulo, Brazil) was obtained from local drugstores. Discarded soft-drink 3. Results and discussion
bottles were used as the source material for acquiring Polyethylene
terephthalate (PET) sheets. The river water sample was obtained from 3.1. Morphological, structural, and electrochemical characterization of
the Paranapanema River in Campina do Monte Alegre (São Paulo, AuNPs-GRA/PET-SPE
Brazil), while the frozen chicken breast meat was acquired from the local
market in the same city. Morphological characterization of the electrode surface and the
All solutions were prepared in ultrapure water, with a resistivity of ≥ modifying dispersion are summarized below. Fig. 1A displays the sur­
18 MΩ•cm, obtained by a Milli-Q purification system (Millipore®). face of PET-SPE observed through scanning electron microscopy (SEM),
revealing a low roughness pattern of the conductive ink. Fig. 1B presents
2.2. Apparatus a bright-field image of the dry 1 mg mL− 1 graphene dispersion obtained
through transmission electron microscopy (TEM), which enables to
All electrochemical measurements were conducted using an Autolab observer detailly the arrangement of the graphene sheets distributed
PGSTAT101 potentiostat/galvanostat, which was managed through along the surface in different sizes, with longitudinal extension ranging
NOVA 2.1.4 software. To examine the morphology of the modified PET- from 2.7 to 8.1 µm, which may be fully open or folded onto themselves.
SPE surface, transmission electron microscopy (TEM) and scanning The inclusion of AuNPs in the graphene dispersion resulted in a homo­
electron microscopy (SEM) were employed, using a transmission geneously structured nanocomposite with well-dispersed nanoparticles

2
L.M.C. Ferreira et al. Food Chemistry 437 (2024) 137698

Fig. 1. (A) SEM images obtained for PET-SPE; (B) bright-field TEM images were obtained for GRA dispersion; (C and D) AuNPs-GRA modifying dispersion at two
magnification levels; (E) X-ray diffractograms of modifying dispersions; (F) respective histogram of the gold particle size distribution; (G) CVs for different stages of
modification on PET-SPE in 0.1 M H2SO4; (H) CVs and (I) Nyquist plots (100 kHz to 1.0 Hz) for different stages of modification on PET-SPE in the presence of 5.0 mM
K4[Fe(CN)6] in 0.1 M KCl solution: unmodified (black), AuNPs film (red), graphene film (green), and graphene decorated with AuNPs (purple). Inset: Randles circuit
simulated for the electrodes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

along the graphene sheets, which can be observed through the dark electrochemical behavior of the AuNPs-GRA/PET-SPE surface in 0.1 M
contrast in Fig. 1C and 1D (higher magnification). H2SO4. The voltammogram reveals two anodic peaks positioned at 0.56
The X-ray diffraction analysis of graphene (Fig. 1E) revealed the V and 1.26 V, accompanied by a cathodic peak at 0.38 V. These peaks are
typical diffraction pattern for this material, displaying a broad peak with attributed to the redox processes involving Au0/3+ within the AuNPs-
a maximum at 24.5◦ , characteristic of the (0 0 2) plane of graphene, GRA/PET-SPE interface. During the anodic scan, AuNPs undergo
indicating that the material has low crystallinity and oxygenated func­ oxidation to Au3+, while the reverse cathodic scan facilitates their
tional groups. This plane appears shifted compared to pure graphite conversion to Au0. The observed gold redox signals remain constant
(typically 26◦ ), indicating a greater interplanar spacing resulting from after 20 voltammetric cycles, indicating the stability of AuNPs film and
the stacking of 2D monolayers. The upper spectrum, in red, corresponds their minimal leaching into the solution. The quantification of the
to the AuNPs-GRA nanocomposite, which shows the graphene-related electroactive species on the electrode surface, Γ, was achieved using the
broad peak overlapping with a set of peaks corresponding to AuNPs or equation Γ = Q/nFA, where Q represents the electric charge after
synthesis byproducts. The diffraction angles of 38.14◦ , 44.3◦ , 64.5◦ , and background correction, obtained by integrating the cathodic peak; n is
77.6◦ are assigned to the (1 1 1), (2 0 0), (2 2 0), and (3 1 1) crystal planes the number of electrons; F is Faraday constant, 96,485.34 C mol− 1; and
of the face-centered cubic crystal structure of gold (JCPDS 04–0784), A is the geometric area of AuNPs-GRA/PET-SPE. Q was found to be 4.73
and the remaining sets of peaks are assigned to NaCl (JCPDS 05–0628). × 10− 5C, and the estimated value of Γ was 8.31 × 10− 10 mol cm− 2.
The histogram in Fig. 1F represents the nanoparticle size distribution, Electrochemical characterization of the electrodes was conducted by
showing particle diameters ranging from 4 to 28 nm, with an average of cyclic voltammetry (CV) and electrochemical impedance spectroscopy
10 nm. (EIS). Fig. 1H shows a set of cyclic voltammograms for 5.0 mM ferro­
In Fig. 1G, the cyclic voltammogram illustrates the distinctive cyanide in 0.1 M KCl. The experiment varying the scan rate at the

3
L.M.C. Ferreira et al. Food Chemistry 437 (2024) 137698

presence of the redox probe (see Figure S1) revealed a gradual increment pronounced increase in peak currents and a positive shift in the cathodic
of the electroactive area, resulting in an estimated value 4.2 times higher peak potential (I) and lower ΔE values for reversible redox process.
for the AuNPs-GRA/PET-SPE compared to the unmodified PET-SPE (see Notably, this film revealed the emergence of oxidation process III, sug­
Table S1). Fig. 1I displays the electrochemical impedance spectra gesting a specific type of interaction between the redox products of ROX
recorded for PET-SPE at different stages of modification. The unmodi­ and the 2D graphene network. The incorporation of AuNPs to form the
fied PET-SPE exhibits characteristic peaks for the Fe3+/Fe2+ redox nanocomposite resulted in a voltammogram with similar profile as the
processes with a distance between oxidation and reduction peaks (ΔE) of graphene film, as well as generating a new discrete increase in peak
0.463 V and a charge transfer resistance (RCT) value of 5.36 kΩ, as currents of approximately 15% compared to the previous film.
indicated by the semicircle diameter in the respective Nyquist plot. After Compared to unmodified PET-SPE, this increase is at least 38% for
modification with AuNPs (AuNPs/PET-SPE), these values decrease to process I and a maximum of 74% for process II.
0.412 V and 2.66 kΩ, respectively, while peak current intensities in­ Regarding the observed redox process, the first cycle did not exhibit
crease. These changes suggest that AuNPs favors charge transfer, any redox processes during the forward scan, only a reduction (peak I, as
improve process reversibility, as well as it promotes a discrete increase shown in Figure S2) with a maximum current at − 0.548 V was observed
of electroactive area. This effect is even more pronounced when the upon reversing the scan direction. During the second cycle, oxidation
electrode is modified with a graphene film (GRA/PET-SPE), with a ΔE processes II (0.35 V), III (0.67 V), and IV (0.92 V) were observed, indi­
value of 0.380 V and RCT of 0.908 kΩ, and an 85% amplification of peak cating that their presence during the scan is dependent on the product
current compared to the base electrode, indicating a significant increase formed in the initial reduction reaction (process I). Following in the
of electroactive area. Finally, the fully modified electrode (AuNPs-GRA/ cathodic direction, the reduction peaks V (0.84 V) and VI (0.226 V) were
PET-SPE) exhibited a ΔE value of 0.063 V, which indicates a decrease of observed, forming reversible pairs with processes IV and II, respectively.
100 mV compared to the base electrode, an RCT of 0.547 kΩ, and a 100% Process III, besides being understood as irreversible, was only observed
signal current amplification. The circuit element properties were ob­ when graphene was incorporated into the electrode modification (see
tained by simulation according to the Randles circuit detailed in Fig. 2H Fig. 2). The assignments of these processes will be subsequently
and presented in Table S1. addressed with the presentation of the proposed mechanism for the ROX
redox reaction.
The variation in the pH of the supporting electrolyte resulted in
3.2. Voltammetric behavior of Roxarsone (ROX) in AuNPs-GRA/PET- changes in the voltammetric profile of ROX, leading to shifts in peak
SPE surface potentials for all redox processes, as depicted in Figure S3. A set of cyclic
voltammograms (2nd cycle) were recorded for AuNPs-GRA/PET-SPE in
Initially, CV experiments were carried out using various modifica­ the presence of ROX at different pHs, ranging from 0 to 5, using two
tions of the PET-SPE in the absence and presence of 1.0 mM ROX. As different electrolyte solutions: HCl (pH 0–1) and 0.05 M Britton-
result, Fig. 2 displays the set of cyclic voltammograms (2nd cycle) Robinson (B-R) buffer + 0.1 M NaCl (pH 2–5). An increase in electro­
recorded for ROX at different PET/SPE electrodes. When using the un­ lyte pH induces a shift in peak potential towards less positive potentials.
modified PET-SPE, only the redox processes I, II, IV, V, and VI are Additionally, the ΔE values for the reversible processes II/VI and IV/V
observed. The incorporation of graphene into the modifying film vary with pH, increasing as the pH increases. Variations in peak current
resulted in a slight increase in capacitive current, and all modifying films intensities are also observed (see Figure S4), with higher values typically
contributed to an overall current increase in all ROX-associated redox observed at pH 1 for most processes, except to reduction process I, which
processes. In the case of the film containing only AuNPs, the current presented minor variations in current intensities with pH changes. At pH
increase was accompanied by a positive shift of most peaks, except for 5, the apparent current increase for process I is primarily attributed to
process I, which presented a and a negative shift (see Table S2). On the electrolyte discharge caused by the peak shift towards more negative
other hand, the film containing only graphene exhibited a more potentials. The peak potential variation shows a linear relationship with
the pH for all observed redox processes. Linear regressions generate
slope values of (62 ± 3), (59 ± 3), (76 ± 2), (70 ± 2), (65 ± 3), and (76
± 3) mV pH− 1 for peaks I, II, II, IV, V, and VI, respectively, slightly
deviating from the Nernstian theoretical value of 59 mV pH− 1, which is
typical for redox reactions involving an equal number of protons and
electrons. The deviations from the theoretical value can be assigned to
eventual polarization events on the pseudo-reference electrode leading
to potential shifts.
Based on the pH effect on the electrochemical response of ROX, it
was possible to propose the redox mechanism for ROX, as illustrated in
Fig. 3. This proposal was based on the results obtained from cyclic
voltammetry experiments and analogous mechanisms reported for 4-
nitrophenol, due the other nitrobenzene derivates (Bharath, Veer­
amani, Chen, Madhu, Manivel Raja, Balamurugan, et al., 2015; Dinesh &
Saraswathi, 2017; Sang, Wang, Wang, Zhao, & Guo, 2014; Y. Tang,
Huang, Liu, Yang, Lu, & Luo, 2013; Tchieno & Tonle, 2018). The
mechanism starts at process I, assigned to the reduction of the nitro
group to hydroxylamine by gaining four electrons and four protons.
From this point, two independent routes are possible: 1) hydroxylamine
is reversibly oxidized to the nitroso group, generating the redox pair II/
VI; 2) In an acidic medium, hydroxylamine undergoes protonation and
loses one water molecule, leading to the formation of benzoquinonei­
Fig. 2. Cyclic voltammograms recorded PET-SPE under different modification
steps: unmodified; modified with gold nanoparticles (AuNPs/PET-SPE); modi­ mine, which is promptly reduced to aminophenol at sweep potentials
fied with graphene (GRA/PET-SPE); and modified with graphene and gold lower than 0.84 V, and subsequently re-oxidized at 0.92 V, forming the
nanoparticles (AuNPs-GRA/PET-SPE) in the presence of 1.0 mM ROX in 0.1 M redox pair IV/V.
HCl. Ei = 0 V, Emax = 1.2 V, Emin = − 0.8 V, scan rate potential of 50 mVs− 1. The transport regime of ROX and its derivate species on the AuNPs-

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L.M.C. Ferreira et al. Food Chemistry 437 (2024) 137698

Fig. 3. Proposed mechanism for ROX redox processes observed via cyclic voltammetry.

GRA/PET-SPE electrode was evaluated by studying the scan rate po­

tential ranging from 10 to 100 mV s− 1, as shown in Figure S5. The set of
+2e− , +2H+ , − H2 O +2e− , +2H+
ROX­NO2 ̅̅̅̅̅̅
̅→ ROX­NO ̅̅̅̅̅→ ROX­NHOH
voltammograms shows an increase in peak current intensity for all redox
From the intercept of Eq. (1), the ks value could be calculated and
processes, exhibiting a linear correlation with the scan rate potential, ν,
found to be 1.30 s− 1.
as well as the logarithm of the current with the logarithm of the scan rate
potential (log I vs. log v), as depicted in Figure S6. By performing linear
regression analysis, two equations were obtained: one relating current 3.3. Voltammetric determination of ROX through preconcentration of
(I) to the scan rate potential (ν), and the other relating the logarithm of nitro group reduction
the current (log I) to the logarithm of the scan rate potential (log ν), such
that the slope associated to nitro group reduction to hydroxylamine Adsorptive characteristic of the redox processes of ROX on the
(process I) was found to be 0.76, indicating that the transport of ROX for AuNPs-GRA/PET-SPE can be applied for preconcentration of the analyte
this specific process is governed by both diffusion and adsorption. In during the reduction step (Process I) and subsequent detection based on
contrast, for the other processes, the slope values are closer to unity, oxidation processes (Processes II/III/IV), ensuring an increase in sensi­
suggesting that the transport for these processes is predominantly gov­ tivity and detection at more favorable potentials, while minimizing the
erned by adsorption. contribution of hydrogen discharge current at negative potentials. For
Additionally, the above-described experiment allowed the acquisi­ analytical purposes, square wave voltammetry (SWV) was chosen for
tion of kinetic parameters for the charge transfer process associated with determination of ROX, due to the reversibility of the process associated
ROX reduction on AuNPs-GRA/PET-SPE electrode, correlating the ob­ with the hydroxylamine-ROX/nitroso-ROX pair (II/VI), which presents
tained data and the Laviron theory for irreversible processes, which an oxidation peak at 0.32 V (see Figure S7). This peak was selected as the
shows a linear dependence between peak potential and the natural primary analytical signal ROX determination, although the response of
logarithm of the scan rate (see Figure S6), according to the Eq. (1) the other two signals (at 0.65 V and 0.86 V) was also evaluated in terms
(Laviron, 1978). of deposition time and concentration variation. The SWV parameters
RT
(
RT
) (
RT
) were investigated, and the optimal condition was achieved under the
EPC = Eo − ln ks + lnν (1) selected parameters: potential step (ΔE = 3 mV), modulation amplitude
αn′F αn′F αn′F
(a = 35 mV) and frequency (f = 35 Hz) according with Table S3.
where α is the charge-transfer coefficient, ks is the heterogeneous Figure S7 also demonstrates the dependence of the oxidation current
electron-transfer rate constant, T is the temperature of the analysis signals on the electrochemical preconcentration process at − 0.6 V,
(298.15 K), F is the Faraday constant (96,485 C mol− 1), R the ideal gas resulting from the reduction of the nitro-ROX group on the surface of the
constant (8.315 J K− 1mol− 1), n’ is the number of electrons transferred in AuNPs-GRA/PET-SPE. In the absence of preconcentration potential, no
the rate-determining step of ROX-NO2 reduction, and E◦ is the standard oxidation peaks are observed. On the other hand, as the duration of
potential. The linear correlation was found and expressed by EPC = potential application increases, the observed signals become more
(–0.614 ± 0.008) + (–0.031 ± 0.002) ln ν. The value of E◦ was obtained prominent, as exemplified by the 30 and 60-second intervals. The
by the linear coefficient of Ep vs Ip plot (− 0.465 V) and α was deduced by deposition time interval for electrochemical preconcentration of ROX
Eq. (2) (Compton & Banks, 2018). was optimized along with the selection of parameters for the SWV
technique, according to the criteria presented in Table S3 for the
⃒ ⃒
⃒EPC − E1/2 ⃒ = 0.0477 (2) oxidation signal at 0.32 V.
α Under the optimized experimental and instrumental conditions,
The correspondent value of α was 0.46, and it was utilized to analytical curves were constructed for ROX concentration ranging from
determine n’ through the slope of Eq. (1). Here, n’ was approximately 0.5 µM to 400 µM. These curves exhibited a linear response in two
1.8 (~2 electrons), as expected for nitrobenzyl group reduction, which concentration intervals for ROX concentration (CROX): from 0.5 µM to
can be split in two processes involving two electrons each (Mu, Rozen­ 20 µM, for the peak at 0.32 V, as depicted in Fig. 4A, and from 20 µM to
dal, Rabaey, & Keller, 2009). 400 µM (see Figure S8). The other two peaks presented narrower con­
centration range in the first interval (1.0 µM to 20 µM for peak at 0.65 V,

5
L.M.C. Ferreira et al. Food Chemistry 437 (2024) 137698

Fig. 4. (A) Square-wave voltammograms for AuNPs-GRA/PET-SPE in 0.1 M HCl containing increasing concentrations of ROX: 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 and
20.0 µM (B) respective calibration plots. SWV parameters: ΔE = 3 mV, a = 35 mV and f = 35 Hz.

and from 2.0 µM to 20 µM for the peak at 0.86 V). The I vs CROX plots are Thus, for the accurate determination of ROX in these samples, an in­
shown in Fig. 4B, which generated the following equations for the lower ternal calibration method must be employed to overcome the matrix
CROX range: effect. Using these calibration curves, the values of the sensitivity, limit
8 of detection (LOD) and limit of quantification (LOQ) for ROX de­
Ip(0.32V) = (8.0 ± 4.0)10− + (0.265 ± 0.010)×CROX, R2 = 0.995 terminations in the sample were estimated. These values are summa­
Ip(0.65V) = (− 7.0 ± 6.0)10− 8
+ (0.080 ± 0.009)×CROX, R2 = 0.945 rized in Table 1, which also presents a comparison with previous
electrochemical methodologies. The samples recovery analysis carried
7
Ip(0.86V) = (− 2.4 ± 0.5)10− + (0.240 ± 0.009)×CROX, R2 = 0.993 out based on standard addition curves in the supporting electrolyte (0.1
M HCl) and in samples enriched with the analyte at a concentration of 5
μM and resulted in the percentage recovery values of 104% for the river
water sample and 89.4% for the diluted chicken purge sample.
3.4. Matrix effect and determination of ROX in food and environmental
The precision of the AuNPs-GRA/PET-SPE was evaluated by testing it
samples
with a 10 µM ROX solution. Repeatability was assessed in two ways: an
intra-day assay (n = 10) and an inter-day assay (n = 5), both using a
A study was conducted to assess the matrix effect of river water and
single modified electrode. Additionally, we examined electrode repro­
diluted chicken purge samples by addition of ROX standard solutions in
ducibility by employing ten different modified electrodes, all featuring
the range from 0.5 to 20 µM. The obtained calibration curves are shown
the same ROX concentration. The resulting voltammograms can be
in Figure S9, along with the respective equations obtained through
observed in Figure S10, and the relative standard deviation (RSD) values
linear regression: I(0.32V) = (2.0 ± 2.0)10− 8 + (0.256 ± 0.003) × CROX,
for current measurements of the anodic peak at 0.38 V were determined
R2 = 0.997 for river water, and I(0.32V) = (2.5 ± 1.0)10− 8 + (0.163 ±
as follows: 4.05% for intra-day repeatability, 10.8% for inter-day
0.003) × CROX, R2 = 0.997 for chicken meat purge. These results
repeatability, and 8.50% for electrode reproducibility. The high RSD
revealed a pronounced matrix effect in the chicken purge sample,
and potential peak shift (50 mV) over the inter-day measurements were
leading to a sensitivity decrease from 0.265 µA/µM to 0.163 µA/µM,
assigned to memory effect due strong adsorption of ROX-derived redox
corresponding to a reduction of approximately 40%, mainly due to the
products. These results demonstrate the satisfactory reproducibility and
high protein concentration in the sample. Meanwhile, the river water
repeatability of the proposed sensor.
sample showed only a slight sensitivity decrease of 3.4%, but it was
statistically different from the standard curve at a 95% confidence level.

Table 1
Comparison of analytical features for ROX determination.
1 2
Electrode Technique Sample Linear range (µM) Sensitivity µA µM− cm− LOD µM LOQ µM Reference
2
Amberlite XAD-4/ DPV poultry feed and litter 2 – 100 11.4 0.53 1.76 (Waris, et al., 2016)
MCPME1
RL-La2(MoO4)3)/SPCE3 DPV chicken/beef 0.025 – 2650 13.4 0.012 n.i.* (Vinoth, Govindasamy,
Wang, Alothman, & Alshgari,
2021)
serum (Chen, Rajaji, Chen,
WS2 NRs/N-rGO4/SPCE DPV urine 0.1 – 443 14.7 0.075 0.1 Chinnapaiyan, &
pharmaceutical samples Ramalingam, 2019)
human urine (Mani Govindasamy, Rajaji,
SrWO4NPs/GrO/ GCE5 CV 0.035 – 1816 9.98 0.022 n.i* Wang, Chang, Ramalingam, &
chicken extract
Chan, 2020)
0.1 M HCl 1.35 0.057 0.19
AuNPs-GRA/PET-SPE SWV river water 0.5 – 20 1.31 0.060 0.20 this work
chicken purge 0.83 0.097 0.32
*
not informed; 1MCPME: modified carbon-paste microelectrode; 2DPV: differential pulse voltammetry; 3RL-La2(MoO4)3): roselike lanthanum molybdate; SPCE:
screen-printed carbon electrode; 4WS2 NRs: WS2 nanorods; N-rGO: N-doped reduced graphene oxide; 5SrWO4NPs/GrO: strontium tungstate decorated on graphene
oxide; GCE: glassy carbon electrode.

6
L.M.C. Ferreira et al. Food Chemistry 437 (2024) 137698

4. Conclusions electrode as portable electrochemical sensing platform for sensitive detection of

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Lohedan, H. A. (2019). A screen-printed electrode modified with tungsten disulfide
nanosheets for nanomolar detection of the arsenic drug roxarsone. Mikrochimica
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Investigation, Writing – original draft, Writing – review & editing. Paulo Govindhan, M., & Chen, A. (2015). Simultaneous synthesis of gold nanoparticle/
graphene nanocomposite for enhanced oxygen reduction reaction. Journal of Power
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Marcio F. Bergamini: Conceptualization, Resources, Writing – review Additives, A Major Step toward Reducing the Human and Ecosystem Health Risk
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Declaration of Competing Interest Kangkamano, T., Numnuam, A., Limbut, W., Kanatharana, P., & Thavarungkul, P.
(2017). Chitosan cryogel with embedded gold nanoparticles decorated multiwalled
carbon nanotubes modified electrode for highly sensitive flow based non-enzymatic
The authors declare that they have no known competing financial
glucose sensor. Sensors and Actuators B: Chemical, 246, 854–863.
interests or personal relationships that could have appeared to influence Kokulnathan, T., Rajagopal, V., Wang, T. J., Huang, S. J., & Ahmed, F. (2021).
the work reported in this paper. Electrochemical Behavior of Three-Dimensional Cobalt Manganate with Flowerlike
Structures for Effective Roxarsone Sensing. Inorganic Chemistry, 60(23),
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