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A novel voltammetric sensor based on carbon nanotubes and nanoparticles of antimony tin oxide for the determination of ractopamine
Materials Science and Engineering C 59 (2016) 368–374
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
A novel voltammetric sensor based on carbon nanotubes and nanoparticles of antimony tin oxide for the determination of ractopamine Aysegul Kutluay Baytak, Tugce Teker, Sehriban Duzmen, Mehmet Aslanoglu ⁎ Department of Chemistry, University of Harran, Sanliurfa 63510, Turkey
a r t i c l e
i n f o
Article history: Received 12 August 2015 Received in revised form 1 October 2015 Accepted 10 October 2015 Available online xxxx Keywords: Ractopamine Antimony tin oxide nanoparticles Carbon nanotubes Composite electrode
a b s t r a c t An electrochemical sensor was prepared by the modification of a glassy carbon electrode (GCE) with carbon nanotubes (CNTs) and nanoparticles of antimony tin oxide (ATO). The surface layer was characterized by scanning electron microscopy (SEM), energy dispersive X-ray diffraction method (EDX) and ATR FT-IR spectroscopy. The proposed electrode was assessed in respect to the electro-oxidation of ractopamine. Compared with a bare GCE and a GCE electrode modified with CNTs, the ATONPs/CNTs/GCE exhibited a great catalytic activity towards the oxidation of ractopamine with a well-defined anodic peak at 600 mV. The current response was linear with the concentration of ractopamine over the range from 10 to 240 nM with a detection limit of 3.3 nM. The proposed electrode enabled the selective determination of ractopamine in the presence of high concentrations of ascorbic acid (AA), dopamine (DA) and uric acid (UA). The proposed electrode was successfully applied for the determination of ractopamine in feed and urine samples. The sensitive and selective determination of ractopamine makes the developed method of great interest for monitoring its therapeutic use and doping control purposes. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Ractopamine, a beta-adrenergic agonist drug, is used to improve weight gain, carcass leanness and feed efficiency in livestock by diverting nutrients from fat deposition to muscle tissue production [1–4]. However, ractopamine is prohibited in horse racing because of its abuse as a stimulant. It has also been banned as a feed additive due to potential risks to the cardiovascular and central nervous systems [4–6]. Thus, the determination of ractopamine is important for both monitoring its therapeutic use and doping cases of horses in competitions. A number of analytical methods have been applied for the determination of ractopamine in samples including immunoassay [7,8], GC–MS [9], LC–MS [10,11], HPLC [12,13], fluorescence spectroscopy [14,15], electrophoresis [16], chemiluminescence [17,18] and voltammetric methods [19–23]. However, some of the above techniques are expensive and require time-consuming steps. Electrochemical sensors have widely been used in electroanalysis due to their unique physical and chemical properties such as excellent electrocatalytic activity, good conductivity and high mechanical strength [24–29]. Furthermore, CNTs and nanoparticle modified electrodes showed great performances in terms of increased sensitivity, mass transport, resistance to surface fouling, decreased overpotential and low detection limit [30–37]. Nanoparticles of metal oxides have several advantages including excellent electrocatalytic activity, increased current response ⁎ Corresponding author. E-mail address: [email protected] (M. Aslanoglu).
http://dx.doi.org/10.1016/j.msec.2015.10.030 0928-4931/© 2015 Elsevier B.V. All rights reserved.
and improved voltammetric behaviour [38,39]. A recent study has indicated that the conductivity and sensitivity increased as antimony was doped into a layer of tin oxide [40]. The nanomaterial based sensors provide highly selective and sensitive approach to the analysis of a wide range of drugs since the electrode materials play a critical role in the construction of high-performance sensing platforms for the determination of drug molecules through various analytical principles [41,42]. Furthermore, functional nanomaterials cannot only produce a synergic effect to accelerate the signal transduction but also amplify biorecognition events for drug sensing [43]. For example, the selectivity is sometimes limited at conventional electrodes due to the interference from the other redox active molecules such as ascorbic acid (AA) and uric acid (UA) which may undergo oxidation at similar potentials to the target drug molecule in biological samples [44–46]. The most important method to overcome such problems is to modify the conventional electrodes with nanomaterials which alter the electrode kinetics of species so that the oxidation potential of the target drug becomes shifted from that required to oxidize the interfering molecules [46–48]. In this study, a novel composite electrode based on carbon nanotubes and nanoparticles of antimony tin oxide has been prepared for the voltammetric determination of ractopamine. The proposed voltammetric sensor exhibited strong catalytic activity towards the oxidation of ractopamine. It also provided a very suitable and effective procedure for the sensitive and selective determination of ractopamine as the modification of GCEs with CNTs and nanoparticles of ATO improved peak separation from interfering compounds such as AA, DA and UA, and improved the detection limit.
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Scheme 1. A schematic illustration of the proposed electrode.
2. Experimental
preparation of solutions. Solutions were deoxygenated by purging nitrogen prior to measurements.
2.1. Chemicals 2.2. Apparatus Ractopamine, AA, DA and UA were obtained from Sigma-Aldrich (St Louis, USA). Chloroform was purchased from Merck (Darmstadt, Germany). Carbon nanotubes (CNTs) and antimony tin oxide nanoparticles were obtained from US-Nano, USA. Stock solutions were prepared with 0.1 M phosphate buffer at pH 7.0. Ultrapure water was used for the
An Autolab potentiostat (Ecochemie, The Netherlands) was used for voltammetric measurements. A three-electrode system was used: a glassy carbon electrode as working electrode [3 mm in diameter (BASi, USA)], a Pt wire counter electrode and a Ag/AgCl reference electrode.
Fig. 1. SEM images of CNTs/GCE (A) and ATONPs/CNTs/GCE (B).
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Fig. 2. EDX analysis of ATONPs/CNTs/GCE.
2.3. Preparation of modified electrodes GCEs were polished with 1 μm and 0.3 μm alumina slurries, sonicated for 5 min in ethanol and rinsed with ultrapure water. GCEs were activated in 0.1 M PBS at pH 7.0 using cyclic voltammetry over the potential range from −1.0 to +1.0 V at a scan rate of 100 mV/s. Prior to the modification, CNTs were functionalized in concentrated HClO4 + HNO3 (3:7, v:v) for 5 h in an ultrasonic bath. Then, CNTs were filtered and washed with water and dried in air. 1 mg of functionalized CNTs and 0.1 mg of ATO nanoparticles were then dispersed in 5 mL of chloroform and sonicated in an ultrasonic bath for 30 min. 5 μL of the composite was cast on the surface of the electrode and then chloroform allowed to evaporate. Afterwards, the resulting electrode denoted as ATONPs/CNTs/GCE was
extensively washed with ultrapure water. The modified electrode (ATONPs/CNTs/GCE) was reactivated in 0.1 M PBS at pH 7.0 by cyclic voltammetry over the range from −0.6 to +0.8 V at a scan rate of 100 mV/s. A schematic illustration of the preparation of the proposed electrode is given in Scheme 1. 3. Results and discussion 3.1. Characterization of modified electrodes The surface layer was characterized by SEM. As given in Fig. 1A, a layer of carbon nanotubes without aggregation was observed indicating that CNTs were homogeneously dispersed on the surface. Fig. 1B
Fig. 3. ATR FT-IR spectra of CNTs (A) and ATONPs-CNTs (B).
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exhibited that ATO nanoparticles were distributed on CNTs. The EDX results exhibited that Sb, Sn, O, C, Pd and Au were the major elements (Fig. 2). However, Pd and Au were obtained from the palladium-gold coating of the electrode during SEM analysis. Fig. 3 shows the ATR FT-IR spectra of CNTs (A) and ATONPs–CNTs (B). In the spectrum of CNTs (Fig. 3A), the absorption band observed at 3415 cm−1 is attributed to the –OH stretching. The absorption peaks observed at 2910 cm−1 and 2850 cm−1 correspond to –CH stretch. The carboxyl characteristic peak is observed at 1635 cm−1. The aromatic –C_C stretch is observed at 1585 cm−1. The absorption peak observed at 1185 cm− 1 can be assigned to the –CO group. In the spectrum of ATONPs–CNTs (Fig. 3B), the absorption observed at 1050, 960 and 850 cm− 1 are due to the vibration of Sn_O, Sn–O and Sb–O [49,50]. The typical Sn–O–Sn vibrations appear in the range of 538–618 cm−1 [50,51]. 3.2. Voltammetric behaviour of ractopamine Fig. 4 exhibits the cyclic voltammograms of ractopamine at bare GCE (A), CNTs/GCE (B) and ATONPs/CNTs/GCE (C) in 0.1 M PBS at pH 7.0. Ractopamine exhibited a poor oxidation peak at 720 mV at bare GCE. However, the GCE modified with CNTs exhibited an improved anodic peak at 630 mV. Furthermore, the voltammetry of ractopamine was highly improved at a GCE modified with a composite of ATONPs and CNTs. The ATONPs/CNTs/GCE exhibited a well-defined anodic peak at 600 mV. It was clearly shown that nanoparticles of ATO provided a high catalytic compared to a bare GCE and CNTs modified GCE. The enhancement in electrocatalytic activity is mainly due to the increase in active surface area and well-distribution of nanoparticles on carbon
Fig. 4. Cyclic voltammograms of 1.5 × 10−7 M ractopamine at GCE (A), CNTs/GCE (B) and ATONPs/CNTs/GCE (C) in 0.1 M PBS at pH 7.0. Scan rate: 50 mV/s.
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nanotubes which provide unique properties, such as high current carrying ability, chemical stability, thermal conductivity and mechanical strength [52–54]. It has also been reported that the enhancement in electrocatalytic activity observed with metal oxide nanoparticles could be attributed to the oxygen vacancies on the nanoparticle [55]. Also, intensive increase in the current response was observed due to the improvement in the electron transfer process of ractopamine and the larger area of the composite layer. Cyclic voltammograms of ractopamine were then recorded at various sweep rates in order to investigate the mechanisms responsible for its oxidation at ATONPs/CNTs/GCE (Fig. 5A). The peak potential, Ep, shifted to more positive values on increasing the sweep rate which confirmed the irreversibility of the process. The current response (Ipa) of ractopamine was proportional to the scan rate (v). Also, log of current response against log of sweep rate showed slope close to 1, suggesting that ractopamine oxidation at ATONPs/CNTs/GCE was a surface-controlled process. The αn value for the oxidation of ractopamine was determined to calculate the number of electrons. The Tafel plot was determined using the following equation.
RT Epa ¼ E þ αn F 0
0
RTk ln αn F
!
þ
RT lnv αn F
As shown in Fig. 4B, the Epa of ractopamine is proportional to the logarithm of sweep rate with a slope of 0.02352 indicating a two-
Fig. 5. (A) Cyclic voltammograms of 1.0 × 10−7 M ractopamine at ATONPs/CNTs/GCE in 0.1 M PBS at pH 7.0. Scan rates: a) 20 mV/s; b) 30 mV/s; c) 40 mV/s; d) 50 mV/s; e) 60 mV/s; f) 70 mV/s; g) 80 mV/s; h) 90 mV/s; and i) 100 mV/s. (B) A plot of peak potentials of ractopamine vs the logarithm of scan rates.
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where Ep is peak potential, E0 is formal potential, m is the number of protons and n is the number of electrons involved in the electrode reaction. The slope of Epa versus pH is 64 mV/pH indicating that the proportion of electrons and protons involved in the reaction is 1e−/1H+ at over the range of pH 4–9 (Fig. 6B). Experimental data indicated that the oxidation process involves a two electron transfer accompanying by two protons. The proposed electrode reaction of ractopamine at ATONPs/CNTs/GCE is given in Scheme 2. 3.4. Calibration equation Fig. 7A shows the square wave voltammograms of various concentrations of ractopamine at ATONPs/CNTs/GCE in 0.1 M PBS at pH 7.0. The current response of ractopamine was linear with its concentration over the range from 10 to 240 nM (Fig. 7B). The linear regression equation was Ipa (μA) = 0.0056 + 0.03942C (μM) with a correlation coefficient of 0.9923. The detection limit was 3.3 nM (The limit of detection, LOD, is based on 3Sb/m where Sb is the standard deviation of the blank signal and m is the slope of the regression equation). The analytical parameters of a number of electrodes for the determination of ractopamine are given in Table 1. The parameters obtained in this study clearly indicate that the proposed electrode is sensitive for the determination of ractopamine as the composite of nanoparticles of ATO and CNTs provides a lower detection limit when compared to the literature values [27,31–33]. 3.5. Detection of ractopamine in the presence of AA, DA and UA
Fig. 6. (A) Cyclic voltammograms of 1.5 × 10−7 M ractopamine at ATONPs/CNTs/GCE in 0.1 M PBS at various pH values. Scan rate: 50 mV/s. (B) A plot of peak potential of ractopamine vs pH.
electron transfer process since the electron transfer coefficient α is approximately 0.5 in a totally irreversible electrode process [56]. 3.3. The effect of pH Fig. 6A shows the effect of the pH value on peak potential of ractopamine at ATONPs/CNTs/GCE. The Epa of ractopamine shifted in the negative direction with increasing pH. The shift in Epa with pH refers to a proton transfer in the oxidation. Based on the following equation, the number of electrons and protons were identical as m/n is close to 1.
Ep ¼ E0
59m pH n
Fig. 8 exhibits CVs of various concentrations of ractopamine in the presence of 25 mM, 0.4 nM DA and 25 mM UA. The current response of ractopamine was proportional to its concentration indicating that the responses of AA, DA and UA had no influence on oxidation of ractopamine. The experimental results showed that higher concentrations of interfering molecules do not interfere with the determination of ractopamine. This indicated that the proposed voltammetric sensor could easily be performed for the quantification of ractopamine in the presence of AA, DA and UA. In other words, the results suggest that the proposed electrode is selective for the determination of ractopamine. The results showed that the proposed electrode system could easily be applied for detecting ractopamine in samples. 3.6. Reproducibility and stability The reproducibility of ATONPs/CNTs/GCE was excellent as the relative standard deviation (RSD) of 10 successive scans was 2.5% for 100 nM ractopamine. Also, 25 cycles of scanning in 0.1 M PBS in the potential range 0.0–1.0 V could regenerate clean background CV curves and the modified electrode was ready for the next experiment or storage in 0.1 M PBS. Furthermore, the peak current decreased only by 5% over 2 weeks for storage in 0.1 M PBS. 3.7. Determination of ractopamine in horse urine samples The voltammetric determination of ractopamine at ATONPs/CNTs/ GCE in horse urine was referred to the regression equation. The results
Scheme 2. The proposed electrode reaction of ractopamine.
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Fig. 8. Cyclic voltammograms of increasing concentrations of ractopamine in the presence of 25 mM AA, 0.4 mM DA and 25 mM UA at ATONPs/CNTs/GCE in 0.1 M PBS at pH 7.0. Ractopamine concentrations: 0.0 nM; 50 nM; 100 nM; 150 nM; and 220 nM. Scan rate: 50 mV/s.
were obtained by ATONPs/CNTs/GCE for the analysis of feed samples. The recoveries and RSD (%) values obtained ractopamine indicate that the ATONPs/CNTs/GCE system is also accurate and precise for the determination of ractopamine in feed samples.
4. Conclusions
Fig. 7. (A) Square wave voltammograms of various concentrations of ractopamine at ATONPs/CNTs/GCE in 0.1 M PBS at pH 7.0. Frequency: 22 Hz. Step potential: 100 mV/s. Amplitude: 50 mV/s. Ractopamine concentrations: 0.0 nM; 10 nM; 20 nM; 40 nM; 50 nM; 60 nM; 70 nM; 80 nM; 100 nM; 120 nM; 150 nM; 180 nM; 220 nM; and 240 nM. (B) A plot of peak currents against the concentrations of ractopamine.
obtained using the proposed electrode is summarised in Table 2. The values of RSD (%) and recoveries obtained for ractopamine confirm that the ATONPs/CNTs/GCE system enables precise quantification of ractopamine in urine samples of racing horses. The accuracy of the proposed electrode was also assured by the analysis of feed samples. The quantification of ractopamine in feed samples has also been referred to the linear equation. Table 3 shows the results of analysis of the feed formulation containing 40 mg/kg ractopamine using the proposed voltammetric sensor. Mean recoveries between 98.7% and 101.2%
Electrochemical determination of ractopamine in horse urine was carried out using a glassy carbon electrode modified with a composite of CNTs and nanoparticles of ATO. Compared with a naked GCE and CNTs/GCE, the ATONPs/CNTs/GCE exhibited a well-defined oxidation peak for ractopamine at 600 mV as well as a remarkable enhancement in current response. The current response of ractopamine was linear with its concentration over the range from 10 to 240 nM with a detection limit of 3.3 nM. High concentrations of AA, DA and UA did not interfere with the selective determination of ractopamine. The proposed procedure exhibited excellent sensitivity and selectivity. The ATONPs/CNTs/GCE was successfully applied for the determination of ractopamine in horse urine and feed samples. The proposed sensor provided a number of combined advantageous in comparison to other methods such as response time, increased sensitivity, high selectivity, decreased overpotential, high effective surface area, catalysis, enhancement of mass transport and low detection limit.
Table 2 The results of recoveries of ractopamine in horse urine using the proposed electrode. Sample 1
2 Table 1 The analytical parameters of a number of electrodes.
Spiked (μg/L)
Found (μg/L)
Recovery%
R.S.D.%
3.01 15.05 60.20 3.01 15.05 60.20
2.95 ± 0.09 15.20 ± 0.42 62.13 ± 1.55 2.98 ± 0.10 15.23 ± 0.46 62.31 ± 1.43
98.0 101.0 103.2 98.5 101.2 103.5
3.2 2.8 2.5 3.4 3.0 2.3
Mean ± standard deviation (n = 5). Electrode
Linear range (μM)
Detection limit (nM)
Reference
Poly/taurine)/ZrO2/GCE Poly(aminothiophenol/Au RTNPs OMC/GCE MWCNT/GCE GO/GCE CNTs-NF/GCE MWCNT-MIM/SPE ATONPs/CNTs/GCE
1–28 2.5–1500 0.1–10 0.085–8 0.148–5.92 0.074–2.96 0.05–33.15 0.02–0.20 0.01–0.24
150 117 74 60 59 56 50 6 3.3
[31] [32] [33] [20] [25] [24] [35] [27] This work
Table 3 Determination of ractopamine in feed formulations. Content (mg)
Added (mg)
Expected (mg)
Found (mg)
Recovery%
R.S.D.%
40
– 10 20
40 50 60
39.48 ± 1.26 50.6 ± 1.52 59.76 ± 2.49
98.7 101.2 99.6
3.2 3.0 2.5
Mean ± standard deviation (n = 5).
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
A novel voltammetric sensor based on carbon nanotubes and nanoparticles of antimony tin oxide for the determination of ractopamine Aysegul Kutluay Baytak, Tugce Teker, Sehriban Duzmen, Mehmet Aslanoglu ⁎ Department of Chemistry, University of Harran, Sanliurfa 63510, Turkey
a r t i c l e
i n f o
Article history: Received 12 August 2015 Received in revised form 1 October 2015 Accepted 10 October 2015 Available online xxxx Keywords: Ractopamine Antimony tin oxide nanoparticles Carbon nanotubes Composite electrode
a b s t r a c t An electrochemical sensor was prepared by the modification of a glassy carbon electrode (GCE) with carbon nanotubes (CNTs) and nanoparticles of antimony tin oxide (ATO). The surface layer was characterized by scanning electron microscopy (SEM), energy dispersive X-ray diffraction method (EDX) and ATR FT-IR spectroscopy. The proposed electrode was assessed in respect to the electro-oxidation of ractopamine. Compared with a bare GCE and a GCE electrode modified with CNTs, the ATONPs/CNTs/GCE exhibited a great catalytic activity towards the oxidation of ractopamine with a well-defined anodic peak at 600 mV. The current response was linear with the concentration of ractopamine over the range from 10 to 240 nM with a detection limit of 3.3 nM. The proposed electrode enabled the selective determination of ractopamine in the presence of high concentrations of ascorbic acid (AA), dopamine (DA) and uric acid (UA). The proposed electrode was successfully applied for the determination of ractopamine in feed and urine samples. The sensitive and selective determination of ractopamine makes the developed method of great interest for monitoring its therapeutic use and doping control purposes. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Ractopamine, a beta-adrenergic agonist drug, is used to improve weight gain, carcass leanness and feed efficiency in livestock by diverting nutrients from fat deposition to muscle tissue production [1–4]. However, ractopamine is prohibited in horse racing because of its abuse as a stimulant. It has also been banned as a feed additive due to potential risks to the cardiovascular and central nervous systems [4–6]. Thus, the determination of ractopamine is important for both monitoring its therapeutic use and doping cases of horses in competitions. A number of analytical methods have been applied for the determination of ractopamine in samples including immunoassay [7,8], GC–MS [9], LC–MS [10,11], HPLC [12,13], fluorescence spectroscopy [14,15], electrophoresis [16], chemiluminescence [17,18] and voltammetric methods [19–23]. However, some of the above techniques are expensive and require time-consuming steps. Electrochemical sensors have widely been used in electroanalysis due to their unique physical and chemical properties such as excellent electrocatalytic activity, good conductivity and high mechanical strength [24–29]. Furthermore, CNTs and nanoparticle modified electrodes showed great performances in terms of increased sensitivity, mass transport, resistance to surface fouling, decreased overpotential and low detection limit [30–37]. Nanoparticles of metal oxides have several advantages including excellent electrocatalytic activity, increased current response ⁎ Corresponding author. E-mail address: [email protected] (M. Aslanoglu).
http://dx.doi.org/10.1016/j.msec.2015.10.030 0928-4931/© 2015 Elsevier B.V. All rights reserved.
and improved voltammetric behaviour [38,39]. A recent study has indicated that the conductivity and sensitivity increased as antimony was doped into a layer of tin oxide [40]. The nanomaterial based sensors provide highly selective and sensitive approach to the analysis of a wide range of drugs since the electrode materials play a critical role in the construction of high-performance sensing platforms for the determination of drug molecules through various analytical principles [41,42]. Furthermore, functional nanomaterials cannot only produce a synergic effect to accelerate the signal transduction but also amplify biorecognition events for drug sensing [43]. For example, the selectivity is sometimes limited at conventional electrodes due to the interference from the other redox active molecules such as ascorbic acid (AA) and uric acid (UA) which may undergo oxidation at similar potentials to the target drug molecule in biological samples [44–46]. The most important method to overcome such problems is to modify the conventional electrodes with nanomaterials which alter the electrode kinetics of species so that the oxidation potential of the target drug becomes shifted from that required to oxidize the interfering molecules [46–48]. In this study, a novel composite electrode based on carbon nanotubes and nanoparticles of antimony tin oxide has been prepared for the voltammetric determination of ractopamine. The proposed voltammetric sensor exhibited strong catalytic activity towards the oxidation of ractopamine. It also provided a very suitable and effective procedure for the sensitive and selective determination of ractopamine as the modification of GCEs with CNTs and nanoparticles of ATO improved peak separation from interfering compounds such as AA, DA and UA, and improved the detection limit.
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Scheme 1. A schematic illustration of the proposed electrode.
2. Experimental
preparation of solutions. Solutions were deoxygenated by purging nitrogen prior to measurements.
2.1. Chemicals 2.2. Apparatus Ractopamine, AA, DA and UA were obtained from Sigma-Aldrich (St Louis, USA). Chloroform was purchased from Merck (Darmstadt, Germany). Carbon nanotubes (CNTs) and antimony tin oxide nanoparticles were obtained from US-Nano, USA. Stock solutions were prepared with 0.1 M phosphate buffer at pH 7.0. Ultrapure water was used for the
An Autolab potentiostat (Ecochemie, The Netherlands) was used for voltammetric measurements. A three-electrode system was used: a glassy carbon electrode as working electrode [3 mm in diameter (BASi, USA)], a Pt wire counter electrode and a Ag/AgCl reference electrode.
Fig. 1. SEM images of CNTs/GCE (A) and ATONPs/CNTs/GCE (B).
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Fig. 2. EDX analysis of ATONPs/CNTs/GCE.
2.3. Preparation of modified electrodes GCEs were polished with 1 μm and 0.3 μm alumina slurries, sonicated for 5 min in ethanol and rinsed with ultrapure water. GCEs were activated in 0.1 M PBS at pH 7.0 using cyclic voltammetry over the potential range from −1.0 to +1.0 V at a scan rate of 100 mV/s. Prior to the modification, CNTs were functionalized in concentrated HClO4 + HNO3 (3:7, v:v) for 5 h in an ultrasonic bath. Then, CNTs were filtered and washed with water and dried in air. 1 mg of functionalized CNTs and 0.1 mg of ATO nanoparticles were then dispersed in 5 mL of chloroform and sonicated in an ultrasonic bath for 30 min. 5 μL of the composite was cast on the surface of the electrode and then chloroform allowed to evaporate. Afterwards, the resulting electrode denoted as ATONPs/CNTs/GCE was
extensively washed with ultrapure water. The modified electrode (ATONPs/CNTs/GCE) was reactivated in 0.1 M PBS at pH 7.0 by cyclic voltammetry over the range from −0.6 to +0.8 V at a scan rate of 100 mV/s. A schematic illustration of the preparation of the proposed electrode is given in Scheme 1. 3. Results and discussion 3.1. Characterization of modified electrodes The surface layer was characterized by SEM. As given in Fig. 1A, a layer of carbon nanotubes without aggregation was observed indicating that CNTs were homogeneously dispersed on the surface. Fig. 1B
Fig. 3. ATR FT-IR spectra of CNTs (A) and ATONPs-CNTs (B).
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exhibited that ATO nanoparticles were distributed on CNTs. The EDX results exhibited that Sb, Sn, O, C, Pd and Au were the major elements (Fig. 2). However, Pd and Au were obtained from the palladium-gold coating of the electrode during SEM analysis. Fig. 3 shows the ATR FT-IR spectra of CNTs (A) and ATONPs–CNTs (B). In the spectrum of CNTs (Fig. 3A), the absorption band observed at 3415 cm−1 is attributed to the –OH stretching. The absorption peaks observed at 2910 cm−1 and 2850 cm−1 correspond to –CH stretch. The carboxyl characteristic peak is observed at 1635 cm−1. The aromatic –C_C stretch is observed at 1585 cm−1. The absorption peak observed at 1185 cm− 1 can be assigned to the –CO group. In the spectrum of ATONPs–CNTs (Fig. 3B), the absorption observed at 1050, 960 and 850 cm− 1 are due to the vibration of Sn_O, Sn–O and Sb–O [49,50]. The typical Sn–O–Sn vibrations appear in the range of 538–618 cm−1 [50,51]. 3.2. Voltammetric behaviour of ractopamine Fig. 4 exhibits the cyclic voltammograms of ractopamine at bare GCE (A), CNTs/GCE (B) and ATONPs/CNTs/GCE (C) in 0.1 M PBS at pH 7.0. Ractopamine exhibited a poor oxidation peak at 720 mV at bare GCE. However, the GCE modified with CNTs exhibited an improved anodic peak at 630 mV. Furthermore, the voltammetry of ractopamine was highly improved at a GCE modified with a composite of ATONPs and CNTs. The ATONPs/CNTs/GCE exhibited a well-defined anodic peak at 600 mV. It was clearly shown that nanoparticles of ATO provided a high catalytic compared to a bare GCE and CNTs modified GCE. The enhancement in electrocatalytic activity is mainly due to the increase in active surface area and well-distribution of nanoparticles on carbon
Fig. 4. Cyclic voltammograms of 1.5 × 10−7 M ractopamine at GCE (A), CNTs/GCE (B) and ATONPs/CNTs/GCE (C) in 0.1 M PBS at pH 7.0. Scan rate: 50 mV/s.
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nanotubes which provide unique properties, such as high current carrying ability, chemical stability, thermal conductivity and mechanical strength [52–54]. It has also been reported that the enhancement in electrocatalytic activity observed with metal oxide nanoparticles could be attributed to the oxygen vacancies on the nanoparticle [55]. Also, intensive increase in the current response was observed due to the improvement in the electron transfer process of ractopamine and the larger area of the composite layer. Cyclic voltammograms of ractopamine were then recorded at various sweep rates in order to investigate the mechanisms responsible for its oxidation at ATONPs/CNTs/GCE (Fig. 5A). The peak potential, Ep, shifted to more positive values on increasing the sweep rate which confirmed the irreversibility of the process. The current response (Ipa) of ractopamine was proportional to the scan rate (v). Also, log of current response against log of sweep rate showed slope close to 1, suggesting that ractopamine oxidation at ATONPs/CNTs/GCE was a surface-controlled process. The αn value for the oxidation of ractopamine was determined to calculate the number of electrons. The Tafel plot was determined using the following equation.
RT Epa ¼ E þ αn F 0
0
RTk ln αn F
!
þ
RT lnv αn F
As shown in Fig. 4B, the Epa of ractopamine is proportional to the logarithm of sweep rate with a slope of 0.02352 indicating a two-
Fig. 5. (A) Cyclic voltammograms of 1.0 × 10−7 M ractopamine at ATONPs/CNTs/GCE in 0.1 M PBS at pH 7.0. Scan rates: a) 20 mV/s; b) 30 mV/s; c) 40 mV/s; d) 50 mV/s; e) 60 mV/s; f) 70 mV/s; g) 80 mV/s; h) 90 mV/s; and i) 100 mV/s. (B) A plot of peak potentials of ractopamine vs the logarithm of scan rates.
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where Ep is peak potential, E0 is formal potential, m is the number of protons and n is the number of electrons involved in the electrode reaction. The slope of Epa versus pH is 64 mV/pH indicating that the proportion of electrons and protons involved in the reaction is 1e−/1H+ at over the range of pH 4–9 (Fig. 6B). Experimental data indicated that the oxidation process involves a two electron transfer accompanying by two protons. The proposed electrode reaction of ractopamine at ATONPs/CNTs/GCE is given in Scheme 2. 3.4. Calibration equation Fig. 7A shows the square wave voltammograms of various concentrations of ractopamine at ATONPs/CNTs/GCE in 0.1 M PBS at pH 7.0. The current response of ractopamine was linear with its concentration over the range from 10 to 240 nM (Fig. 7B). The linear regression equation was Ipa (μA) = 0.0056 + 0.03942C (μM) with a correlation coefficient of 0.9923. The detection limit was 3.3 nM (The limit of detection, LOD, is based on 3Sb/m where Sb is the standard deviation of the blank signal and m is the slope of the regression equation). The analytical parameters of a number of electrodes for the determination of ractopamine are given in Table 1. The parameters obtained in this study clearly indicate that the proposed electrode is sensitive for the determination of ractopamine as the composite of nanoparticles of ATO and CNTs provides a lower detection limit when compared to the literature values [27,31–33]. 3.5. Detection of ractopamine in the presence of AA, DA and UA
Fig. 6. (A) Cyclic voltammograms of 1.5 × 10−7 M ractopamine at ATONPs/CNTs/GCE in 0.1 M PBS at various pH values. Scan rate: 50 mV/s. (B) A plot of peak potential of ractopamine vs pH.
electron transfer process since the electron transfer coefficient α is approximately 0.5 in a totally irreversible electrode process [56]. 3.3. The effect of pH Fig. 6A shows the effect of the pH value on peak potential of ractopamine at ATONPs/CNTs/GCE. The Epa of ractopamine shifted in the negative direction with increasing pH. The shift in Epa with pH refers to a proton transfer in the oxidation. Based on the following equation, the number of electrons and protons were identical as m/n is close to 1.
Ep ¼ E0
59m pH n
Fig. 8 exhibits CVs of various concentrations of ractopamine in the presence of 25 mM, 0.4 nM DA and 25 mM UA. The current response of ractopamine was proportional to its concentration indicating that the responses of AA, DA and UA had no influence on oxidation of ractopamine. The experimental results showed that higher concentrations of interfering molecules do not interfere with the determination of ractopamine. This indicated that the proposed voltammetric sensor could easily be performed for the quantification of ractopamine in the presence of AA, DA and UA. In other words, the results suggest that the proposed electrode is selective for the determination of ractopamine. The results showed that the proposed electrode system could easily be applied for detecting ractopamine in samples. 3.6. Reproducibility and stability The reproducibility of ATONPs/CNTs/GCE was excellent as the relative standard deviation (RSD) of 10 successive scans was 2.5% for 100 nM ractopamine. Also, 25 cycles of scanning in 0.1 M PBS in the potential range 0.0–1.0 V could regenerate clean background CV curves and the modified electrode was ready for the next experiment or storage in 0.1 M PBS. Furthermore, the peak current decreased only by 5% over 2 weeks for storage in 0.1 M PBS. 3.7. Determination of ractopamine in horse urine samples The voltammetric determination of ractopamine at ATONPs/CNTs/ GCE in horse urine was referred to the regression equation. The results
Scheme 2. The proposed electrode reaction of ractopamine.
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Fig. 8. Cyclic voltammograms of increasing concentrations of ractopamine in the presence of 25 mM AA, 0.4 mM DA and 25 mM UA at ATONPs/CNTs/GCE in 0.1 M PBS at pH 7.0. Ractopamine concentrations: 0.0 nM; 50 nM; 100 nM; 150 nM; and 220 nM. Scan rate: 50 mV/s.
were obtained by ATONPs/CNTs/GCE for the analysis of feed samples. The recoveries and RSD (%) values obtained ractopamine indicate that the ATONPs/CNTs/GCE system is also accurate and precise for the determination of ractopamine in feed samples.
4. Conclusions
Fig. 7. (A) Square wave voltammograms of various concentrations of ractopamine at ATONPs/CNTs/GCE in 0.1 M PBS at pH 7.0. Frequency: 22 Hz. Step potential: 100 mV/s. Amplitude: 50 mV/s. Ractopamine concentrations: 0.0 nM; 10 nM; 20 nM; 40 nM; 50 nM; 60 nM; 70 nM; 80 nM; 100 nM; 120 nM; 150 nM; 180 nM; 220 nM; and 240 nM. (B) A plot of peak currents against the concentrations of ractopamine.
obtained using the proposed electrode is summarised in Table 2. The values of RSD (%) and recoveries obtained for ractopamine confirm that the ATONPs/CNTs/GCE system enables precise quantification of ractopamine in urine samples of racing horses. The accuracy of the proposed electrode was also assured by the analysis of feed samples. The quantification of ractopamine in feed samples has also been referred to the linear equation. Table 3 shows the results of analysis of the feed formulation containing 40 mg/kg ractopamine using the proposed voltammetric sensor. Mean recoveries between 98.7% and 101.2%
Electrochemical determination of ractopamine in horse urine was carried out using a glassy carbon electrode modified with a composite of CNTs and nanoparticles of ATO. Compared with a naked GCE and CNTs/GCE, the ATONPs/CNTs/GCE exhibited a well-defined oxidation peak for ractopamine at 600 mV as well as a remarkable enhancement in current response. The current response of ractopamine was linear with its concentration over the range from 10 to 240 nM with a detection limit of 3.3 nM. High concentrations of AA, DA and UA did not interfere with the selective determination of ractopamine. The proposed procedure exhibited excellent sensitivity and selectivity. The ATONPs/CNTs/GCE was successfully applied for the determination of ractopamine in horse urine and feed samples. The proposed sensor provided a number of combined advantageous in comparison to other methods such as response time, increased sensitivity, high selectivity, decreased overpotential, high effective surface area, catalysis, enhancement of mass transport and low detection limit.
Table 2 The results of recoveries of ractopamine in horse urine using the proposed electrode. Sample 1
2 Table 1 The analytical parameters of a number of electrodes.
Spiked (μg/L)
Found (μg/L)
Recovery%
R.S.D.%
3.01 15.05 60.20 3.01 15.05 60.20
2.95 ± 0.09 15.20 ± 0.42 62.13 ± 1.55 2.98 ± 0.10 15.23 ± 0.46 62.31 ± 1.43
98.0 101.0 103.2 98.5 101.2 103.5
3.2 2.8 2.5 3.4 3.0 2.3
Mean ± standard deviation (n = 5). Electrode
Linear range (μM)
Detection limit (nM)
Reference
Poly/taurine)/ZrO2/GCE Poly(aminothiophenol/Au RTNPs OMC/GCE MWCNT/GCE GO/GCE CNTs-NF/GCE MWCNT-MIM/SPE ATONPs/CNTs/GCE
1–28 2.5–1500 0.1–10 0.085–8 0.148–5.92 0.074–2.96 0.05–33.15 0.02–0.20 0.01–0.24
150 117 74 60 59 56 50 6 3.3
[31] [32] [33] [20] [25] [24] [35] [27] This work
Table 3 Determination of ractopamine in feed formulations. Content (mg)
Added (mg)
Expected (mg)
Found (mg)
Recovery%
R.S.D.%
40
– 10 20
40 50 60
39.48 ± 1.26 50.6 ± 1.52 59.76 ± 2.49
98.7 101.2 99.6
3.2 3.0 2.5
Mean ± standard deviation (n = 5).
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