ORIGINAL PAPER

Electrochemical immunosensing using a nanostructuredfunctional platform for determination of α-zearalanol

Matías Regiart & Marco A. Seia & Germán A. Messina &

Franco A. Bertolino & Julio Raba

Received: 9 June 2014 /Accepted: 1 September 2014# Springer-Verlag Wien 2014

Abstract We describe an electrochemical immunosensor forthe determination of the growth promoter α-zearalanol inbovine serum. The sensing scheme is based on a nanocom-posite consisting of gold nanoparticles electrodeposited onmulti-walled carbon nanotubes that were modified with poly(vinylpyridine) through in-situ polymerization. The electrode-position of the gold nanoparticles enlarges the surface avail-able for immobilization of antibodies against α-zearalanol.The nanocomposite film was characterized by scanning elec-tron microscopy, energy dispersive X-ray spectroscopy, andcyclic voltammetry. The calibration plot has a linear responsein the concentrations range from 0.05 to 50 ng mL−1, and thedetection limit is 16 pg mL−1. The time required for analysis is12 min only which compares quite favorably with the time(90 min) required by the conventional ELISA. The methodexhibits good selectivity, stability and reproducibility for de-tecting α-zearalanol in the livestock production.

Keywords Electrochemical . Immunosensor .

Nanocomposite .α-zearalanol . Bovine serum

Introduction

α-zearalanol (ZEN) is a non-steroidal estrogenic agonist gen-erated by chemical reduction of the mycotoxin zearalenone,

which is produced by Fusarium spp and it is commonly foundin contaminated grain products [1, 2]. ZEN belongs to theresorcylic acid lactones family [3], and it has been widely usedas growth promoter in the livestock production [4]. Previousstudies have shown their potential risk factor for breast cancer,with strong and prolonged endocrine-disruptive effects inhumans at very low concentrations [5]. Compounds identifiedas endocrine disrupting compounds (EDCs) are members ofdifferent groups of chemicals, including drugs, pesticides,industrial products, alkylphenols, synthetic steroids, and soon [6]. ZEN is a member of EDCs and its administration as agrowth promoter has been banned in the European Unionsince 1985 [7].

Different methods have been reported for the determinationof EDCs in biological samples, such as capillaryelectrophoresis-amperometric detection [8], liquidchromatography-mass spectrometry [9], ultra high perfor-mance liquid chromatography-mass spectrometry [10], gaschromatography–mass spectrometry [11] and biosensors[12]. Immunoassay techniques, which are based on the highlyspecific molecular recognition of antigens by antibodies, havebecome important analytical methods in clinical, biochemical,pharmaceutical and environmental fields [13]. In recent years,electrochemical immunosensors have gained interest becauseof their high sensitivity, low detection limit, fast analysis andlow cost of the instrumentation [14–16]. Screen-printing isone of the most promising approaches towards simple, rapidand inexpensive production of biosensors. Screen-printedtechnology has advantages of design flexibility, process auto-mation, good reproducibility, a wide choice of materials [17].

Since the discovery of carbon nanotubes (CNTs) in 1991by Ijima [18], there have emerged several modificationmethods with the aim of use them as immobilization platform.Most of these strategies alter the structural and functionalproperties of CNTs due to the extensive and strong chemicaltreatments that they are subjected [19]. For this reason, the use

Electronic supplementary material The online version of this article(doi:10.1007/s00604-014-1355-x) contains supplementary material,which is available to authorized users.

M. Regiart :M. A. Seia :G. A. Messina : F. A. Bertolino (*) :J. Raba (*)INQUISAL-CONICET, Departamento de Química, UniversidadNacional de San Luis, Chacabuco, 917, D5700BWS San Luis,Argentinae-mail: bertolin@unsl.edu.are-mail: jraba@unsl.edu.ar

Microchim ActaDOI 10.1007/s00604-014-1355-x

of polymerization methods not only maintain the nanotubesproperties such as high chemical and mechanical stability,high conductivity, high specific surface area [20], but alsoincorporate a high biocompatibility, hydrophilicity, stabilityand permeability of the new material. These types of strategiesused the polymerization of amonomer in the presence of CNTs,forming a uniform framework that shows the same physico-chemical properties in each point of the framework [21].

In this way, the multi-walled carbon nanotubes (MWCNTs)modified with poly (vinylpyridine) (PVP) offer a large specif-ic surface area with high chemical stability and mechanicalstrength. MWCNTs improve the conductivity of the preparednanocomposite film [22]. The chemical functionalization ofMWCNTs is important not only to increase their dispersion,but also to facilitate their interaction with organic and biolog-ical molecules [23]. Moreover, the porous three dimensionalfilms could provide a high accessible surface area and goodbiocompatible microenvironment for immobilization of anti-bodies [24]. In recent years, noble metal nanoparticles, withextraordinary conductivity, large surface-to-volume ratio andbiocompatibility, have been extensively employed for devel-oping novel electrochemical sensing platforms and improvingtheir performances [25]. Based on the foregoing, the electro-deposition of gold nanoparticles (AuNPs) on these platformshave many properties such as high active surface for antibod-ies immobilization, facilitate the electron transfer due to theirgood conductivity, excellent film-forming ability, high waterpermeability, good adhesion, nontoxicity and remarkable bio-compatibility [26–28].

To the best of our knowledge, no study involving theconstruction and characterization of a nanostructured func-tional platform on portable screen-printed carbon electrode(SPCE) for the immunocapture of ZEN and its subsequentdetermination by square-wave voltammetry (SWV) has beenreported. Therefore, in this article, we present and discuss anovel nanocomposite based on MWCNTs-PVP with AuNPselectrodeposited, applied to the sensitive and selective deter-mination of ZEN in bovine serum.

Materials and methods

Reagents

All reagents used were of analytical reagent grade. ZEN, bo-vine serum albumin (BSA, 98 %), 3-mercaptopropionic acid(MPA), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide(EDC), N-hydroxysuccinimide (NHS), HAuCl4 0.01 %, azobis(isobutyronitrile) (AIBN), multi-walled carbon nanotubes(90 % purity, 10–15 nm external diameter, 2–6 nm internaldiameter, 0.1–10 μm length), 4-vinylpyridine, potassium ferro-cyanide, potassium ferricyanide and N,N-dimethylformamide(DMF) were purchased from Sigma Aldrich (St. Louis, MO,

USA, http://www.sigmaaldrich.com). Polytetrafluoroethylenemembrane (PTFE) (0.2 μm) was obtained from Millipore(Billerica, MA, USA, http://www.millipore.com). Rabbit anti-ZEN antibody was purchased from CER GROUP (Marloie,Belgium, http://www.cergroupe.be/en). All other reagents andsolvents employed were of analytical grade and they were usedwithout further purifications. All solutions were prepared withultra-high-quality water obtained from a Barnstead Easy pureRF compact ultra-pure water system.

Equipment

Scanning electron micrographs were obtained using a LEO1450VP scanning electron microscope (SEM, http://labmem.unsl.edu.ar). The elemental composition of the nanostructuredfilm was determined by energy dispersive X-ray spectroscopy(EDS, http://labmem.unsl.edu.ar) using a Genesis 2000spectrometer. Cyclic voltammetry (CV) and square wavevoltammetry (SWV) measurements were performed using aBAS 100 B/W (electrochemical analyzer Bioanalytical Sys-tem, West Lafayette, IN, http://www.basinc.com). A SPCEmade up of three electrodes was used. A silver ink aspseudo-reference electrode, a graphite ink as auxiliary elec-trode and, a graphite ink circular (Ø=3 mm) with and withoutmodifications as working electrode were used for all themeasurements (DropSens, Llanera, Asturias, Spain,http://www.dropsens.com). All solutions and reagents wereconditioned to 25±1 °C before the experiment, using alaboratory water bath (Vicking Mason II, Vicking SRL,Argentina, http://www.vicking.com.ar). Absorbance wasdetected by a Bio-Rad Benchmark microplate reader (Japan,http://www.bio-rad.com) and Beckman DU 520 general UV/vis spectrophotometer (http://www.beckmancoulter.com). AllpH measurements were made with an Orion expandable ionanalyzer Model EA 940 equipped with a glass combinationelectrode (Orion Research Inc., http://www.bioesanco.com.ar).

Construction of the AuNPs/MWCNTs-PVP functionalplatform on SPCE

In order to obtain the nanocomposite of MWCNTs polymer-ized with PVP, we used the procedure described by Silva et al.[22] with the following modifications. In a 50 mL flaskcontaining 40 mL of DMF were added 50 mg of MWCNTsunder nitrogen atmosphere and the mixture was stirred for15 min. Afterwards, 70 mg of AIBN and 10 mL of 4-vinylpyridine were added. The solution was placed in athermostated oil bath at 80 °C under stirring for 48 h.After this period, the mixture was cooled to room tem-perature and diluted to 250 mL with DMF. The superna-tant was centrifuged for 6 h and vacuum-filtered througha 0.20 μm PTFE membrane.

M. Regiart et al.

To obtain the dispersion of MWCNTs-PVP nanocompos-ite, the following procedure was employed: 6 mg of thenanocomposite was dispersed in 1 mL DMF and ultra soni-cated for 2 h in order to obtain a homogeneous suspension. Analiquot of 2.5 μL of this dispersion was added to the SPCEand then, the electrode was dried at room temperature.

For the electrodeposition procedure of AuNPs, the SPCEwas immersed into 0.01 % HAuCl4 solution containing0.1 mol L−1 KCl (prepared in doubly distilled water anddeaerated by bubbling with nitrogen) as supporting electro-lyte. After that, a constant potential value of −0.4 V wasapplied for 30 s. Then, the modified electrode was rinsed withdoubly distilled water and dried carefully with pure nitrogengas. Finally, the AuNPs/MWCNTs-PVP/SPCEwas optimizedand characterized by SEM, EDS and CV.

Preparation of anti-ZEN-AuNPs/MWCNTs-PVP/SPCE

After obtaining the AuNPs/MWCNTs-PVP/SPCE, it wasplaced in contact with MPA 0.04 mol L−1 in EtOH/H2O(75:25, v/v) for 15 h at room temperature. At this point, the -SH group of MPA reacted with the surface of AuNPs previ-ously electrodeposited, leaving as a result free -COOH groups,which were activated by rinsing in a solution containing EDCand NHS in 0.01 mol L−1 phosphate buffered saline (PBS)pH 7.20, and then evaporated to dryness. After that the elec-trode was rinsed with doubly distilled water and dried withpure nitrogen gas. Finally, a solution of anti-ZEN 1/500 in0.01 mol L−1 PBS pH 7.20 was placed on the surface of theAuNPs/MWCNTs-PVP/SPCE working electrode and incu-bated overnight at 4 °C. After that, the immunosensor wasrinsed with 0.01 mol L−1 PBS pH 7.20 and stored at 4 °C inthe same buffer when not in use. Scheme 1 shows the proce-dure of the immunosensor preparation.

Procedure for immunocapture and detection of ZEN

This method was applied to the determination of ZEN inbovine serum using a micro-volume electrochemical cell.

Prior to the analysis of each serum sample, the unspecificbinding of the modified AuNPs/MWCNTs-PVP/SPCE work-ing electrode was blocked by 2 min treatment of 500 μL 1 %BSA in a 0.01 mol L−1 PBS pH 7.20 and then washed severaltimes with 500 μL of 0.01 mol L−1 PBS pH 7.20. The serumsamples were first diluted 50-fold with 0.01 mol L−1 PBSpH 7.20 and then 200 μL was added to the immunosensor for5 min. In this step, the ZEN present in the sample bindsimmunologically to the anti-ZEN antibodies. Then, theimmunosensor was washed three successive times with0.01 mol L−1 PBS pH 7.20. Finally, the electrode was placedin 100 μL of desorption buffer (0.1 mol L−1 glycine-HClpH 2.0) where ZEN was desorbed on the modified electrodesurface and it was oxidized at +1.12 V. A standardcurve for the immunosensor was produced followingour protocol with a series of standards that coveredthe relevant range (0–100 ngmL−1) suppliedwith the ELISAtest kit. When not in use, the immunosensor was stored in0.01 mol L−1 PBS pH 7.20 at 4 °C. Scheme 2 shows theprocedure for immunocapture and detection of ZEN.

Results and discussion

Characterization of the AuNPs/MWCNTs-PVP/SPCE

Morphologies of SPCE, MWCNTs/SPCE MWCNTs-PVP/SPCE and AuNPs/MWCNTs-PVP/SPCE nanocompositefilms were investigated by SEM. Figure 1a shows the baregraphite carbon surface. Figure 1b reveals that MWCNTs arewell distributed on the SPCE surface and, Fig. 1c shows thechange in the morphology ofMWCNTs due to the presence ofPVP, indicating that PVP was successfully polymerized overthe nanomaterial. In addition, Fig. 1d reveals that the electro-deposition of AuNPs onto the nanocomposite film not mod-ified the morphology of this material. The three-dimensionalstructure of AuNPs/MWCNTs-PVP nanocomposite film pro-vides a suitable surface for anti-ZEN antibodies

AuNPs

Anti-α-zearalanol

MWCNTs-PVP SPCE

HAuCl4Scheme 1 Procedure for the anti-ZEN-AuNPs/MWCNTs-PVP/SPCE preparation

Electrochemical immunosensing using a nanostructured platform

immobilization and a conductive pathway for electron-trans-fer. The elemental composition of the nanocomposite wasdetermined by EDS. Figure 1e shows the three peaks of

interest, at 0.3, 2.3 and 9.8 keV, corresponding to the C andAu atoms, respectively.

Electrochemical responseof the AuNPs/MWCNTs-PVP/SPCE

Figure 2 shows the CVof modified and unmodified SPCE for1 mmol L−1 ferrocyanide/ferricyanide redox couple solution,containing 0.1 mol L−1 KCl solution from −0.2 to + 0.8 Vat ascan rate of 75 mV s−1. Well-defined CVs and characteristicdiffusion-controlled redox process were observed at the SPCEsurface (black line). The red line illustrates the voltammetriceffect of the redox couple on the MWCNTs-PVP film. Whenthe dispersion of MWCNTs-PVP was deposited on the elec-trode surface, the peak current was larger than the observed forbare SPCE, indicating that the nanocomposite improved theconductivity and increased the active surface area of theelectrode.When AuNPs/MWCNTs-PVPwas used (blue line),the highest peak current was obtained, due to the large surfacearea of AuNPs and MWCNTs. The average value of surfacearea for AuNPs/MWCNTs-PVP/SPCE was 12.65 (±0.15)×10−2 cm2 (n=6) according to the Randles-Sevick equation,that is Ip=2.69×10

5 AD1/2 n3/2 ν1/2 C.

Desorptionbuffer

anti-ZEN-AuNPs/MWCNTs-PVP/SPCE

SWV

α-zearalanol reduced

α-zearalanol oxidized

E (V)

Scheme 2 Procedure forimmunocapture and detectionof ZEN

300 nm a) nm

300 nm 300 nm

b)

c) d)

e)

300

Fig. 1 Morphology characterization of AuNPs/MWCNTs-PVP/SPCEnanocomposite. The morphologies of a) SPCE, b) MWCNTs/SPCE c)MWCNTs-PVP/SPCE and d) AuNPs/MWCNTs-PVP/SPCE nanocom-posite films were investigated by SEM. e) The elemental composition ofthe nanocomposite was determined by EDS

-0,2 0,0 0,2 0,4 0,6 0,8-200

-150

-100

-50

0

50

100

150

200 SPCE MWCNTs-PVP/SPCE AuNPs/MWCNTs-PVP/SPCE

I (

A)

E (V)

μΔ

Fig. 2 Electrochemical characterization of the different modified elec-trodes. CVs were performed in 1 mmol L−1 K3[Fe(CN)6]/K4[Fe(CN)6] in0.1 mol L−1 KCl (pH 6.50) solution from −0.2 to +0.8 Vat a scan rate of75 mV s−1

M. Regiart et al.

Electrochemical behavior of ZENon AuNPs/MWCNTs-PVP/SPCE

After performing the electrochemical characterization of mod-ified electrodes, the ZEN behavior was studied with modifiedelectrodes by CV as shown in Fig. 3. The figure shows atypical voltammogram of 1 mmol L−1 ZEN in 0.1 mol L−1

glycine-HCl pH 2.0 solution as supporting electrolyte (scanrate = 75 mV s−1; T=25±1 °C). The cyclic voltammogramshowed a single anodic peak at Epa = +1.12 V for the potentialrange +0.1 to +1.4 V, which corresponded to the one-electron

transfer of ZEN. When the sweep was performed in thereverse direction, no reduction peak was observed, showingthat the ZEN oxidation is an irreversible process at the surfaceelectrode under the experimental conditions. As can be seen inFig. 3, the modification of the electrode surface with AuNPs/MWCNTs-PVP nanocomposite increased the oxidation peakcurrent and hence, the sensitivity of the method.

Election of the electroanalytical technique

The immunocapture of ZEN on the anti-ZEN-AuNPs/MWCNTs-PVP/SPCE was an efficient method for the deter-mination of very low levels of ZEN in real samples. The ZENbound to the immunosensor could be desorbed and directlymeasured in 0.1 mol L−1 glycine-HCl buffer at pH 2.0. TheSWV has several advantages compared to other electroana-lytical techniques such as, high sensitivity, great speed ofanalysis, low consumption of the electroactive species andreduction of the problems with the poisoning of the electrodesurface. For this reason, the electrochemical sensing ofZEN in bovine serum was carried out using SWV bydirect measurement of its oxidation peak after desorptionof the immunosensor.

Optimization of experimental conditions

In order to perform the electrochemical sensing of ZEN inbovine serum, many variables that affect the electrochemical

0,2 0,4 0,6 0,8 1,0 1,2 1,4-5

0

5

10

15

20

25

30 AuNPs/MWCNTs-PVP/SPCE MWCNTs-PVP/SPCE SPCE BLANK

I (

A)

E (V)

Δμ

Fig. 3 Electrochemical behavior of 1 mmol L−1 ZEN in 0.1 mol L−1

glycine-HCl (pH 2.0) with different modified electrodes

Fig. 4 Optimization ofexperimental conditions. a)The amount of polymerdeposited on the electrode, b)the electrodeposition time ofAuNPs, c) the electrodepositionpotential of AuNPs, and d) thecalibration curve for ZEN wasobtained in a linear range from0.05 to 50 ng mL−1, with adetection limit of 16 pg mL−1

Electrochemical immunosensing using a nanostructured platform

response must be analyzed. The SWV fixed electrochemicalparameters to carry out the optimization of these relevantvariables were: step E = 0.010 V, S.W. amplitude = 0.050 V,S.W. frequency = 10 Hz, samples per point = 256, studiedpotential range = +0.1 to +1.4 V, sensitivity = 1×10−5 AV−1

using anti-ZEN-AuNPs/MWCNTs-PVP/SPCE as workingelectrode. Regarding to the ZEN concentration, a standardsolution of 25 ng mL−1 was employed in all studies.

The amount of polymer deposited on the graphite electrodesurface was studied between 0.5 and 4 μL. The current re-sponse was linearly increased from 0.5 to 2 μL and thenremaining constant. Then, we choose 2.5 μL for the optimalamount of polymer deposited on the electrode surface(Fig. 4a). Another parameter analyzed was the electrodeposi-tion time of the AuNPs which was studied in the range of 10–60 s. The results showed that the signal increased within thefirst 30 s and then remains constant. Therefore, 30 s was

chosen as the optimal electrodeposition time (Fig. 4b). Finallywe analyzed the electrodeposition potential of the AuNPs onthe electrode surface between −0.1 and −0.6 V being observedan increase in the signal to −0.6 V. Hence we used −0.4 V(Fig. 4c).

The influence of the desorption buffer on the modifiedelectrode response was tested in two different buffer solutions(glycine-HCl and citrate-HCl) in the concentration of0.1 mol L−1 at pH 2.0, obtaining higher Ipa responses inglycine-HCl solution (475 nA against 403 nA of citrate-HClsolution). In this sense, glycine-HCl buffer solution was cho-sen for further studies. The results obtained for the measure-ments carried out in different concentrations of glycine-HClbuffer (0.01–0.50 mol L−1) showed that Ipa valuesreached a maximum when 0.1 mol L−1 was employedand then decreased for solutions with higher concentrations(Online Resource 1). Finally, the pH of glycine-HCl buffer wasinvestigated in the pH range from 1 to 6, obtaining decreasedIpa values when the pH increased (Online Resource 2). Thus,0.1mol L−1 glycine-HCl buffer solution at pH 2.0 was selectedas optimum condition for further analyses.

Analytical parametersof anti-ZEN-AuNPs/MWCNTs-PVP/SPCE

The calibration curve for the electrochemical sensing of ZENwas obtained by using the electrochemical immunosensorunder optimal experimental conditions. The change of oxida-tion peak current response of the immunosensor was found tobe proportional to the ZEN concentration in a linear rangefrom 0.05 to 50 ngmL−1, with a detection limit of 16 pgmL−1.The data were analyzed by linear regression least-squares fitmethod. The calibration graph was described by the

Table 1 Determination of ZEN in bovine serum by both methods

Samplea Methods

VISb EIAc

BS 1d 2.79±0.11e 2.81±0.15

BS 2 10.51±0.38 10.32±0.24

BS 3 22.15±0.26 21.97±0.41

BS 4 32.83±0.33 33.28±0.25

a Bovine serum samplesb Voltammetric immunosensorc Enzyme immunoassayd Bovine Serum and controle Mean of five determinations ± S.D

Table 2 Comparison with other papers involving modified electrodes with different electrochemical techniques for ZEN determination

Modification Electrode Technique Sample Linear range(ng mL−1)

LOD (pg mL−1)

CS-PtCo-Ab/graphene nanosheets [13] GCEa CVb Urine 0.05–5 13

Na-Mont-TH-HRP-Ab2/nanoporous gold films-Ab1 [14] GCE Ac Liver 0.01–12 3

Graphene sheets-nickel hexacyanoferratenanocomposites-Ab [15]

GEd A Liver 0.05–10 6

Gold nanoparticles/poly (vinylpyridine)-multiwalledcarbon nanotubes [this work]

SPCEe SVWf Serum 0.05–50 16

a Glassy carbon electrodeb Cyclic voltammetryc Amperometryd Gold electrodee Screen printed carbon electrodef Square wave voltammetry

M. Regiart et al.

calibration equation ΔIpa (nA) = 3.12+19.30 CZEN with acorrelation coefficient for this plot of 0.996, where ΔIpa isthe difference between current of the blank and samples(Fig. 4d). The ZEN quantification was directly carried outover diluted samples, and not matrix effects were observed.

Now a day, the possibility of re-use immunosensorsis an important characteristic in biosensors field. Thisimmunosensor was regenerated by simply treating withdesorption solution for 5 min to dissociate theantibodies-antigen complex. The long-term stability ofimmunosensor was investigated over a 20 day period,stored at 4 °C. The current response to 25 ng mL−1

ZEN maintained about 91 % of the original value afterthe storage period. The good stability may be attributedto the fact that the immunosensor could provide abiocompatible microenvironment for the antibodiesimmobilization.

The reproducibility of the immunosensor was studiedby intra- and inter-assay. The intra-assay precision wasevaluated by assaying the ZEN level for five determi-nations of one sample in the same day. The intra-assayCV of this method was 4.53 % at ZEN concentrationsof 25 ng mL−1. The inter-assay precision was evaluatedby determining the ZEN level in one sample along aweek. The inter-assay CV was 5.92 % at the ZENconcentration of 25 ng mL−1, showing an acceptablereproducibility.

In order to evaluate the immunosensor response, itwas applied to the determination of ZEN in five bovineserum samples under the conditions previously de-scribed and the results were compared by enzymeimmunoassay. The positive samples were analyzed byour method, which revealed similar concentrations ofZEN in all of them (Table 1). Another important pa-rameter was the analysis time that was only 12 minagainst the 90 min that takes the enzyme immunoassaytechnique.

Table 2 summarized and compared the most relevantarticles related to modified electrodes with different elec-trochemical techniques for ZEN determination. It is im-portant to highlight that this is the first electrochemicalimmunosensor for ZEN determination in bovine serumusing a portable SPCE modified with AuNPs/MWCNTs-PVP nanocomposite. Our detection system also used avery sensitive electroanalytical technique such as SWV,combined with anti-ZEN antibodies that provide a highselectivity to the electrochemical sensor. Moreover, inthis method we measure the oxidation peak current ofZEN, and the current response was proportional with theconcentration in the sample. Finally, the electrochemicalmethod showed an appropriate linear range and LOD forsensing of growth promoter in bovine serum relative toother electrochemical techniques for ZEN determination.

Conclusion

This article described the development of an electrochemicalimmunosensor for α-zearalanol detection by the constructionof a nanostructured functional platform. The AuNPs/MWCNTs-PVP nanocomposite was synthesized using softconditions and the resulting material showed a good stability.On one hand, an important contribution of this article wasdemonstrate that even if low conductivity polymers wereformed, the contribution of each nanomaterial was clear toenhance the electronic communication and increase the re-sponse of the electrode. On the other hand, the developedmethod showed many advantages like portability, low cost,wide linear range, accuracy with excellent LOD. Anotherimportant parameter was the analysis time that was only12min against the 90min that takes the enzyme immunoassaytechnique. Finally, this method could be a very promisinganalytical tool for the direct determination of growth promoterin the livestock production, ensuring safety and quality offood, as well as consumer’s health.

Acknowledgments The authors wish to thank the financial supportfrom Universidad Nacional de San Luis (UNSL), Instituto de Químicade San Luis (INQUISAL), Consejo Nacional de InvestigacionesCientíficas y Técnicas (CONICET) and Servicio Nacional de Sanidad yCalidad Agroalimentaria (SENASA).

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