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Glassy carbon electrode modified with gold nanoparticles for ractopamine and metaproterenol sensing
Chemical Physics Letters 574 (2013) 83–88
Contents lists available at SciVerse ScienceDirect
Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett
Glassy carbon electrode modified with gold nanoparticles for ractopamine and metaproterenol sensing Jiahua Duan, Dawei He ⇑, Wenshuo Wang ⇑, Yongchuan Liu, Hongpeng Wu, Yongsheng Wang, Ming Fu Key Laboratory of Luminescence and Optical Information, Ministry of Education, Institute of Optoelectronic Technology, Beijing Jiaotong University, Beijing 100044, China
a r t i c l e
i n f o
Article history: Received 2 March 2013 In final form 23 April 2013 Available online 2 May 2013
a b s t r a c t In this Letter, the gold nanoparticles (AuNPs) were used as an enhanced material for selective detection of ractopamine and metaproterenol with electrochemical methods. The morphology and size of gold nanoparticles were characterized by scanning electron microscopy and absorption spectrum. Meanwhile, the electrical properties of modified glass carbon electrode (GCE) were studied by electrochemical impedance spectroscopy. The electrochemical behaviors of ractopamine and metaproterenol were well explained by PM3 calculated method and cyclic voltammetry. Importantly, the ractopamine and metaproterenol were effectively detected. The detection range has been broadened to (109–105 M) and the detection time has been shortened to a few minutes. Crown Copyright Ó 2013 Published by Elsevier B.V. All rights reserved.
1. Introduction Beta-adrenergic agonists are phenylethanolamine compounds which were applied in therapy for pulmonary disease in humans and animals [1]. In recent years, these illegal food additives were used for livestock farming because of their muscle growing promotion effects and lipid degradation [2]. Meanwhile, many athletes took beta-agonists for triumph due to their anabolic effects including the decreasing fat deposition and increasing lean muscle [3]. However, the consumption of these compounds will throw a serious threat to people’s health due to the long half-life and slow metabolism [4]. In order to guarantee the health of citizens and the justice of sport competitions, the b-agonists have been banned by many governments and International Olympic Committee [5]. Among them, the ractopamine (Rac) and metaproterenol (Meta) were in widely illegal applications and affected the export exchange transaction of edible meat products resulting in huge economic loss. To date, various analytical methods were proposed to selectively detect Rac and Meta, which included high performance liquid chromatography (HPLC) [6], electrochemiluminescence method [7], gas chromatography–mass spectrometry (GC–MS) [8], immunomagnetic proximity ligation assay [9], surface enhanced Raman spectrometry [10] and so on. Although these methods are promising due to their high selectivity and sensitivity, they are expensive and time-consuming because of their complicated separation process and pre-concentrated procedure. Hence, it is significantly crucial to develop a simple, convenient and accurate method for ⇑ Corresponding authors. Fax: +86 10 51688018 (D. He). E-mail addresses: [email protected] (D. He), [email protected] (W. Wang).
detection of Rac and Meta. Compared to those methods, the electrochemical approach has many advantages including time-saving, simplicity, no need for expensive instruments and so on. Given in that these compounds contains electroactive phenolic or resorcinol group, the electrochemical assay could be a potential route for determination of Rac and Meta. Although the electrochemical methods for detection of Rac were proposed by other literatures [11,12], they have high limit (108 M order of magnitude) and narrow linear range (108–106 M). On the other hand, the electrochemical detection of Meta has not been developed in the recent years. Hence, it shows greatly meaningful to develop a new and highly selective electrochemical detection of Rac and Meta. Gold nanoparticles (AuNPs) have drawn great attention due to their unique physical and chemical properties including high conductivity, high specific surface area, peculiar optical properties and so on [13]. Based on these astonishing properties, AuNPs were successfully applied to surface enhanced Raman spectrometry (SERS) [14], photovoltaic devices [15], catalysis [16], imaging and diagnosis for cancer or other disease [17], biosensors [18] and so on. Meanwhile, gold nanoparticles were also used in electrode modification for detection of many compounds including toxic substances such as atenolol [19], rutin [20], heavy metal ions [21], DNA [22], glucose [23], uric acid [24] and so on. However, to the best of our knowledge, there are few methods reported about bio-assay for Rac and Meta based on glassy carbon electrode modified with gold nanoparticles. Herein, we proposed a novel and convenient approach to detect trace amounts of Rac and Meta based on gold nanoparticles modified electrodes through cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The AuNPs solution was directly dropped on the bare glass carbon electrode (GCE) and dried under
0009-2614/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cplett.2013.04.057
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infrared lamp. As confirmed by electrochemical impedance spectroscopy (EIS), the AuNPs modified electrode (AuNPs/GCE) displays better conductivity than bare GCE. The electrochemical behaviors of Rac and Meta were also studied by CV and theoretically explained by PM3 algorithm methods. Furthermore, different concentrations of Rac and Meta were determined by DPV within a few minutes. The linear range was from 109 to 105 M and the limit of detection was 3 1010 M (based on signal-to-noise ratio of 3). More importantly, this method shows promising application for detection of other b-agonists analogs and other compounds containing similar electroactive groups. 2. Material and methods 2.1. Instrumentation All electrochemical measurements were carried out on a CHI 660D electrochemical workstation (Chenhua Instrument, Shanghai, China). A conventional three-electrode system was applied with bare GCE or AuNPs/GCE as the working electrode, a platinum wire as the counter electrode, and a saturated calomel electrode (SCE) as the reference electrode. Scanning electron microscopy (SEM) images of AuNPs were achieved by an S-4800 (Hitachi, Japan) at 15.0 kV. The optical absorption spectra of AuNPs were obtained by a UV-3101 scanning spectrophotometer (Shimadzu, Japan) at room temperature. 2.2. Chemicals and reagents All reagents used in this Letter were of analytical grade and used as received without further purification. Rac hydrochloride and Meta hemisulfate were both purchased from Sigma (USA). As reported elsewhere [25], the stock solutions were prepared by dissolving Rac or Meta in pH 2.0 B–R buffer (Britton–Robinson buffer) solution. The B–R buffer was prepared by 2.875 mL acetic acid, 3.38 mL phosphoric acid, 3.11 g boric acid and deionized water. All solutions were prepared with double distilled water and stored at 4 °C. 2.3. Fabrication of gold nanoparticles
2.5. Assay procedure Different concentrations of Rac and Meta were individually added into pH 2.0 B–R buffer and then accumulated at 0.2 V while stirring the solution. The electrochemical impedance spectroscopy (EIS) experiments were performed in the presence of 10 mM K3Fe(CN)6 as the redox probe in a frequency range from 0.1 Hz to 0.1 MHz. The applied potential was 0.25 V and signal amplitude was 5 mV. CV was carried out for the research of their electrochemical behaviors and DPV was applied for the selective detection of Rac and Meta. The CV curves were recorded from 0.2 to 1.2 V under different scan rates in the presence of 106 M Rac or Meta. Meanwhile, the DPV were performed with different concentrations from 0.8 to 1.2 V and the oxidation peak current at 1.12 V was measured for the detection of Rac and Meta. The pulse amplitude was 50 mV and the pulse width was 200 ms. 3. Results and discussion 3.1. Characterization The morphologies and size of the gold nanoparticles are shown in inset from Figure 1. The mono-dispersed state AuNPs was successfully fabricated as confirmed by SEM. The mean size of spherical nanoparticles is about 40 nm. Furthermore, the optical absorption spectrum of AuNPs is shown in Figure 1. As shown, the intrinsic band at about 528.5 nm corresponds to other reported literature [27] which further indicates the successful fabrication of dispersed AuNPs. The narrow band demonstrates the excellent dispersed state of gold nanoparticles. In order to test the performance of AuNPs, the conducting features are performed by EIS with 10 mM K3Fe(CN)6 as the redox probe. In the high frequency range of Nyquist plot, the AuNPs/ GCE displays a smaller semicircle diameter, relating to the charge transfer resistance, than the bare GCE (Figure 2). This indicates that the modified electrode shows higher electron transfer rate than the bare one, which is significantly meaningful for the bio-assay. In the lower frequency, the inclined portion of the curve (about 45°) is associated with the Warburg impedance, which is responsible for the frequency dependence of ion diffusion from the electrolyte to the electrode surface. As shown, the Warburg impedance of modified electrode (corresponding to the evolution of Z00 as a function
The gold nanoparticles were produced through reduction of HAuCl4 with sodium citrate as previously reported [26]. All glassware used in this fabrication was thoroughly washed in aqua regia (VHCl/VHNO3 = 3/1) and rinsed with doubly distilled water. The fabrication process was as follows: First, 500 lL of HAuCl4 (25.4 mM) was added into 40 mL boiling distilled water under vigorous stirring. Second, 900 lL of trisodium citrate (34 mM) was quickly added into the boiling solution under refluxing for about 15 min to get a wine red solution. Finally, the suspension was cooled down to room temperature under continuous stirring. As confirmed by SEM, the produced spherical AuNPs demonstrated a mean diameter of 40 nm under good dispersed state. 2.4. Preparation of the AuNPs modified GCEs Before modification, the GCEs were respectively polished by 1.0, 0.3 and 0.05 lm aluminum slurry for 5 min, and rinsed thoroughly with doubly distilled water between two polishing process. Next, the polished GCEs were successively sonicated with acetone, ethanol, redistilled water and finally dried under ambient condition. The AuNPs modified GCE were prepared by casting 10 lL of AuNPs solutions onto the surface of bare GCE and dried under infrared lamp.
Figure 1. The UV–vis absorption spectrum of mono-dispersed AuNPs. The resonated wavelength is about 528.5 nm. Inset: SEM micrographs of fabricated AuNPs. The mean size of AuNPs is about 40 nm.
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Figure 2. Electrochemical impedance spectroscopy of bare GCE and AuNPs/GCE for the Faradaic impedance measurements.
Figure 3. The cyclic voltammetry curves of 105 M ractopamine at bare GCE and AuNPs modified GCE. Scan rate: 100 mV/s.
of the inverse of the square root of the frequency) is larger than the one of bare electrode. This phenomenon indicates that the AuNPs on the surface of electrode increases the contact surface area of the ferricyanide ions. All the results demonstrate the successful modification of electrode with AuNPs. Figure 3 shows the CVs of a bare GCE and AuNPs/GCE in 105 M Rac solutions. Due to the good conductivity and large specific surface area of AuNPs, the modified electrode displays obvious oxidation peak of Rac, which cannot be found with the bare electrode. Furthermore, through the electrostatic attractions, the absorption of Rac is enhanced thanks to the opposite charged situation between AuNPs (negative) and Rac (positive). This indicates that the successful modification with AuNPs can effectively enhance the sensitivity of bio-assay for Rac and Meta.
3.2. The electrochemical behavior of Rac and Meta In order to explain the oxidation mechanism of Rac and Meta, the structural optimization and Mulliken charges are calculated by PM3 method. As shown in Figure 4A, the aliphatic hydroxyl group displays the most negative charges in the entire molecular structure of Rac. Meanwhile, the aliphatic hydroxyl group similarly shows the most negative charges in Meta. Hence, we provide primitive explanation to the electrochemical oxidation process as followings (Figure 4B): The main oxidation peak (1.12 V) is probably oxidation of aliphatic hydroxyl group in the Rac or Meta as other literature reported [28]. This process generates radicals and may further combine into dimers which are easier oxidized. So, the low potential oxidation and reduction peak (0.60 V) may
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Figure 4. (A) The optimized structure of ractopamine and metaproterenol with their Mulliken charges. (B) The electrochemical oxidation.
Figure 5. (A) Cyclic voltammograms of 106 M ractopamine at AuNPs/GCE with different scan rates. (B) The peak current at 1.12 V of ractopamine vs. the scan rate.
Figure 6. (A) Cyclic voltammograms of 106 M metaproterenol at AuNPs/GCE with different scan rates. (B) The peak current at 1.12 V of metaproterenol vs. the scan rate.
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Figure 7. (A) Differential pulse voltammograms of different concentrations of ractopamine at AuNPs modified GCE. The pulse period: 0.2 s, amplitude: 50 mV. (B) The calibration curve between logarithm of concentration and the current of peak based on DPV.
Figure 8. (A) Differential pulse voltammograms of different concentrations of metaproterenol at AuNPs modified GCE. The pulse period: 0.2 s, amplitude: 50 mV. (B) The calibration curve between logarithm of concentration and the current of peak based on DPV.
be attributed to the electrochemical response of dimers which need more research. The CV curves of 106 M Rac at AuNPs/GCE under different scan rates are represented in Figure 5A. As shown, the oxidation peak current of Rac is increasing with the adding scan rates. It is found that the oxidation peak currents are proportional to the scan rates (R = 0.98), which indicates a typical surface-controlled electrochemical behavior of Rac (Figure 5B). Meanwhile, the same behavior happens to the Meta as illustrated in Figure 6.
3.3. The determination of Rac and Meta In order to detect Rac selectively, the DPVs are carried out as illustrated in Figure 7A. From Figure 7A, the oxidation peak currents of Rac are increasing with gradual addition of concentrations. A typical linear relationship with a correlation coefficient of 0.98 (Figure 7B) exists between peak currents and logarithm of concentration. The detection range is from 109 to 105 M and the limit of detection is 3 1010 M (S/N = 3). Similarly, the determination of Meta is shown in Figure 8 and the linear range is also from 109 to 105 M. Compared with other methods reported above, this proposed approach has advantages including time-saving (a few minutes), low cost, wide linear range and low detection limit.
4. Conclusion In this Letter, the electrochemical behaviors of Rac and Meta on the AuNPs modified electrode were studied by PM3 method and CV curves. The mechanism of electrochemical process of Rac and Meta was proposed and the surface-controlled electrochemical oxidation was studied. The AuNPs were applied in modification of GCE for detection of Rac and Meta. During the detection, AuNPs with good conductivity, larger specific surface area and negatively charged surface catalyzed the oxidation of Rac and Meta effectively. The linear range of Rac and Meta are from 109 to 105 M and the limit of detection is 3 1010 M (S/N = 3). The high selectivity and convenience of AuNPs modified GCE is promising for the detection of Rac and Meta in clinical applications. Furthermore, this simple sensing platform may be extended to the determination of other b-agonist analogs and bio-compounds with electroactive groups. Acknowledgements The authors gratefully acknowledge the financial support from the National Basic Research Program 973:2011CB932700, 2011CB932703, National Outstanding Youth Science Foundation under Contract No. 60825407, National Natural Science Fund Project under Contract No. 60877025, 61077044, 91123025, Beijing
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Natural Science Fund Project under Contract No. 4132031, and the Fundamental Research Funds for the Central Universities of Beijing Jiaotong University under Contract No. 2012YJS124, 2013JBM097 References [1] W. Meng, J. Wei, X. Luo, F. Zhang, B. Yang, Z. Guo, X. Liang, Anal. Methods 4 (2012) 1163. [2] G. Brambilla et al., Toxicol. Lett. 114 (2000) 47. [3] L. Amendola, C. Colamonici, F. Rossi, F. Botrè, J. Chromatogr. B 773 (2002) 7. [4] G. Mazzanti, C. Daniele, G. Boatto, G. Manca, G. Brambilla, A. Loizzo, Toxicology 187 (2003) 91. [5] A.A.M. Stolker, U.A.T. Brinkman, J. Chromatogr. A 1067 (2005) 15. [6] M. Rambla-Alegre, J. Peris-Vicente, S. Marco-Peiró, B. Beltrán-Martinavarro, J. Esteve-Romero, Talanta 81 (2010) 894. [7] S. Wang, J. Wei, T. Hao, Z. Guo, J. Electroanal. Chem. 664 (2011) 146. [8] L. He, Y. Su, Z. Zeng, Y. Liu, X. Huang, Anim. Feed Sci. Technol. 132 (2007) 316. [9] X. Jiang et al., Analyst 138 (2013) 438. [10] F. Zhai, Y. Huang, C. Li, X. Wang, K. Lai, J. Agric. Food Chem. 59 (2011) 10023. [11] Z. Liu, Y. Zhou, Y. Wang, Q. Cheng, K. Wu, Electrochim. Acta 74 (2012) 139. [12] H. Zhang, Y. Ye, C. Chai, G. Liu, Anal. Lett. 45 (2012) 1736. [13] K. Saha, S.S. Agasti, C. Kim, X. Li, V.M. Rotello, Chem. Rev. 112 (2012) 2739.
[14] P. Wang, D. Zhang, L. Zhang, Y. Fang, Chem. Phys. Lett. 556 (2012) 146. [15] J. Chen, Y. Zou, Y. Li, X. Zhou, J. Zhang, X. Li, X. Xiao, Y. Lin, Chem. Phys. Lett. 460 (2008) 168. [16] M.D. Hughes et al., Nature 437 (2005) 1132. [17] R. Weissleder, Nat. Biotechnol. 19 (2001) 316. [18] S.S. Dasary, U.S. Rai, H. Yu, Y. Anjaneyulu, M. Dubey, P.C. Ray, Chem. Phys. Lett. 460 (2008) 187. [19] S. Pruneanu, F. Pogacean, C. Grosan, E.M. Pica, L.C. Bolundut, A.S. Biris, Chem. Phys. Lett. 504 (2011) 56. [20] M. Liu et al., Biosens. Bioelectron. 41 (2013) 275. [21] Y. Zhang, X. Lu, X. Dong, K. Zhang, Anal. Methods 4 (2012) 3326. [22] N. Wu, W. Gao, X. He, Z. Chang, M. Xu, Biosens. Bioelectron. 39 (2013) 210. [23] J. Li, Z. Yang, Y. Zhang, S. Yu, Q. Xu, Q. Qu, X. Hu, Microchim. Acta 179 (2012) 265. [24] X. Tian, C. Cheng, H. Yuan, J. Du, D. Xiao, S. Xie, M.M.F. Choi, Talanta 93 (2012) 79. [25] L. Liu, H. Pan, M. Du, W. Xie, J. Wang, Electrochim. Acta 55 (2010) 7240. [26] K.C. Grabar, R.G. Freeman, M.B. Hommer, M.J. Natan, Anal. Chem. 67 (1995) 735. [27] J. Zhang, L. Wang, D. Pan, S. Song, F.Y. Boey, H. Zhang, C. Fan, Small 4 (2008) 1196. [28] L. Shen, Z. Li, P. He, Electrochem. Commun. 12 (2010) 876.
Contents lists available at SciVerse ScienceDirect
Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett
Glassy carbon electrode modified with gold nanoparticles for ractopamine and metaproterenol sensing Jiahua Duan, Dawei He ⇑, Wenshuo Wang ⇑, Yongchuan Liu, Hongpeng Wu, Yongsheng Wang, Ming Fu Key Laboratory of Luminescence and Optical Information, Ministry of Education, Institute of Optoelectronic Technology, Beijing Jiaotong University, Beijing 100044, China
a r t i c l e
i n f o
Article history: Received 2 March 2013 In final form 23 April 2013 Available online 2 May 2013
a b s t r a c t In this Letter, the gold nanoparticles (AuNPs) were used as an enhanced material for selective detection of ractopamine and metaproterenol with electrochemical methods. The morphology and size of gold nanoparticles were characterized by scanning electron microscopy and absorption spectrum. Meanwhile, the electrical properties of modified glass carbon electrode (GCE) were studied by electrochemical impedance spectroscopy. The electrochemical behaviors of ractopamine and metaproterenol were well explained by PM3 calculated method and cyclic voltammetry. Importantly, the ractopamine and metaproterenol were effectively detected. The detection range has been broadened to (109–105 M) and the detection time has been shortened to a few minutes. Crown Copyright Ó 2013 Published by Elsevier B.V. All rights reserved.
1. Introduction Beta-adrenergic agonists are phenylethanolamine compounds which were applied in therapy for pulmonary disease in humans and animals [1]. In recent years, these illegal food additives were used for livestock farming because of their muscle growing promotion effects and lipid degradation [2]. Meanwhile, many athletes took beta-agonists for triumph due to their anabolic effects including the decreasing fat deposition and increasing lean muscle [3]. However, the consumption of these compounds will throw a serious threat to people’s health due to the long half-life and slow metabolism [4]. In order to guarantee the health of citizens and the justice of sport competitions, the b-agonists have been banned by many governments and International Olympic Committee [5]. Among them, the ractopamine (Rac) and metaproterenol (Meta) were in widely illegal applications and affected the export exchange transaction of edible meat products resulting in huge economic loss. To date, various analytical methods were proposed to selectively detect Rac and Meta, which included high performance liquid chromatography (HPLC) [6], electrochemiluminescence method [7], gas chromatography–mass spectrometry (GC–MS) [8], immunomagnetic proximity ligation assay [9], surface enhanced Raman spectrometry [10] and so on. Although these methods are promising due to their high selectivity and sensitivity, they are expensive and time-consuming because of their complicated separation process and pre-concentrated procedure. Hence, it is significantly crucial to develop a simple, convenient and accurate method for ⇑ Corresponding authors. Fax: +86 10 51688018 (D. He). E-mail addresses: [email protected] (D. He), [email protected] (W. Wang).
detection of Rac and Meta. Compared to those methods, the electrochemical approach has many advantages including time-saving, simplicity, no need for expensive instruments and so on. Given in that these compounds contains electroactive phenolic or resorcinol group, the electrochemical assay could be a potential route for determination of Rac and Meta. Although the electrochemical methods for detection of Rac were proposed by other literatures [11,12], they have high limit (108 M order of magnitude) and narrow linear range (108–106 M). On the other hand, the electrochemical detection of Meta has not been developed in the recent years. Hence, it shows greatly meaningful to develop a new and highly selective electrochemical detection of Rac and Meta. Gold nanoparticles (AuNPs) have drawn great attention due to their unique physical and chemical properties including high conductivity, high specific surface area, peculiar optical properties and so on [13]. Based on these astonishing properties, AuNPs were successfully applied to surface enhanced Raman spectrometry (SERS) [14], photovoltaic devices [15], catalysis [16], imaging and diagnosis for cancer or other disease [17], biosensors [18] and so on. Meanwhile, gold nanoparticles were also used in electrode modification for detection of many compounds including toxic substances such as atenolol [19], rutin [20], heavy metal ions [21], DNA [22], glucose [23], uric acid [24] and so on. However, to the best of our knowledge, there are few methods reported about bio-assay for Rac and Meta based on glassy carbon electrode modified with gold nanoparticles. Herein, we proposed a novel and convenient approach to detect trace amounts of Rac and Meta based on gold nanoparticles modified electrodes through cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The AuNPs solution was directly dropped on the bare glass carbon electrode (GCE) and dried under
0009-2614/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cplett.2013.04.057
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infrared lamp. As confirmed by electrochemical impedance spectroscopy (EIS), the AuNPs modified electrode (AuNPs/GCE) displays better conductivity than bare GCE. The electrochemical behaviors of Rac and Meta were also studied by CV and theoretically explained by PM3 algorithm methods. Furthermore, different concentrations of Rac and Meta were determined by DPV within a few minutes. The linear range was from 109 to 105 M and the limit of detection was 3 1010 M (based on signal-to-noise ratio of 3). More importantly, this method shows promising application for detection of other b-agonists analogs and other compounds containing similar electroactive groups. 2. Material and methods 2.1. Instrumentation All electrochemical measurements were carried out on a CHI 660D electrochemical workstation (Chenhua Instrument, Shanghai, China). A conventional three-electrode system was applied with bare GCE or AuNPs/GCE as the working electrode, a platinum wire as the counter electrode, and a saturated calomel electrode (SCE) as the reference electrode. Scanning electron microscopy (SEM) images of AuNPs were achieved by an S-4800 (Hitachi, Japan) at 15.0 kV. The optical absorption spectra of AuNPs were obtained by a UV-3101 scanning spectrophotometer (Shimadzu, Japan) at room temperature. 2.2. Chemicals and reagents All reagents used in this Letter were of analytical grade and used as received without further purification. Rac hydrochloride and Meta hemisulfate were both purchased from Sigma (USA). As reported elsewhere [25], the stock solutions were prepared by dissolving Rac or Meta in pH 2.0 B–R buffer (Britton–Robinson buffer) solution. The B–R buffer was prepared by 2.875 mL acetic acid, 3.38 mL phosphoric acid, 3.11 g boric acid and deionized water. All solutions were prepared with double distilled water and stored at 4 °C. 2.3. Fabrication of gold nanoparticles
2.5. Assay procedure Different concentrations of Rac and Meta were individually added into pH 2.0 B–R buffer and then accumulated at 0.2 V while stirring the solution. The electrochemical impedance spectroscopy (EIS) experiments were performed in the presence of 10 mM K3Fe(CN)6 as the redox probe in a frequency range from 0.1 Hz to 0.1 MHz. The applied potential was 0.25 V and signal amplitude was 5 mV. CV was carried out for the research of their electrochemical behaviors and DPV was applied for the selective detection of Rac and Meta. The CV curves were recorded from 0.2 to 1.2 V under different scan rates in the presence of 106 M Rac or Meta. Meanwhile, the DPV were performed with different concentrations from 0.8 to 1.2 V and the oxidation peak current at 1.12 V was measured for the detection of Rac and Meta. The pulse amplitude was 50 mV and the pulse width was 200 ms. 3. Results and discussion 3.1. Characterization The morphologies and size of the gold nanoparticles are shown in inset from Figure 1. The mono-dispersed state AuNPs was successfully fabricated as confirmed by SEM. The mean size of spherical nanoparticles is about 40 nm. Furthermore, the optical absorption spectrum of AuNPs is shown in Figure 1. As shown, the intrinsic band at about 528.5 nm corresponds to other reported literature [27] which further indicates the successful fabrication of dispersed AuNPs. The narrow band demonstrates the excellent dispersed state of gold nanoparticles. In order to test the performance of AuNPs, the conducting features are performed by EIS with 10 mM K3Fe(CN)6 as the redox probe. In the high frequency range of Nyquist plot, the AuNPs/ GCE displays a smaller semicircle diameter, relating to the charge transfer resistance, than the bare GCE (Figure 2). This indicates that the modified electrode shows higher electron transfer rate than the bare one, which is significantly meaningful for the bio-assay. In the lower frequency, the inclined portion of the curve (about 45°) is associated with the Warburg impedance, which is responsible for the frequency dependence of ion diffusion from the electrolyte to the electrode surface. As shown, the Warburg impedance of modified electrode (corresponding to the evolution of Z00 as a function
The gold nanoparticles were produced through reduction of HAuCl4 with sodium citrate as previously reported [26]. All glassware used in this fabrication was thoroughly washed in aqua regia (VHCl/VHNO3 = 3/1) and rinsed with doubly distilled water. The fabrication process was as follows: First, 500 lL of HAuCl4 (25.4 mM) was added into 40 mL boiling distilled water under vigorous stirring. Second, 900 lL of trisodium citrate (34 mM) was quickly added into the boiling solution under refluxing for about 15 min to get a wine red solution. Finally, the suspension was cooled down to room temperature under continuous stirring. As confirmed by SEM, the produced spherical AuNPs demonstrated a mean diameter of 40 nm under good dispersed state. 2.4. Preparation of the AuNPs modified GCEs Before modification, the GCEs were respectively polished by 1.0, 0.3 and 0.05 lm aluminum slurry for 5 min, and rinsed thoroughly with doubly distilled water between two polishing process. Next, the polished GCEs were successively sonicated with acetone, ethanol, redistilled water and finally dried under ambient condition. The AuNPs modified GCE were prepared by casting 10 lL of AuNPs solutions onto the surface of bare GCE and dried under infrared lamp.
Figure 1. The UV–vis absorption spectrum of mono-dispersed AuNPs. The resonated wavelength is about 528.5 nm. Inset: SEM micrographs of fabricated AuNPs. The mean size of AuNPs is about 40 nm.
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Figure 2. Electrochemical impedance spectroscopy of bare GCE and AuNPs/GCE for the Faradaic impedance measurements.
Figure 3. The cyclic voltammetry curves of 105 M ractopamine at bare GCE and AuNPs modified GCE. Scan rate: 100 mV/s.
of the inverse of the square root of the frequency) is larger than the one of bare electrode. This phenomenon indicates that the AuNPs on the surface of electrode increases the contact surface area of the ferricyanide ions. All the results demonstrate the successful modification of electrode with AuNPs. Figure 3 shows the CVs of a bare GCE and AuNPs/GCE in 105 M Rac solutions. Due to the good conductivity and large specific surface area of AuNPs, the modified electrode displays obvious oxidation peak of Rac, which cannot be found with the bare electrode. Furthermore, through the electrostatic attractions, the absorption of Rac is enhanced thanks to the opposite charged situation between AuNPs (negative) and Rac (positive). This indicates that the successful modification with AuNPs can effectively enhance the sensitivity of bio-assay for Rac and Meta.
3.2. The electrochemical behavior of Rac and Meta In order to explain the oxidation mechanism of Rac and Meta, the structural optimization and Mulliken charges are calculated by PM3 method. As shown in Figure 4A, the aliphatic hydroxyl group displays the most negative charges in the entire molecular structure of Rac. Meanwhile, the aliphatic hydroxyl group similarly shows the most negative charges in Meta. Hence, we provide primitive explanation to the electrochemical oxidation process as followings (Figure 4B): The main oxidation peak (1.12 V) is probably oxidation of aliphatic hydroxyl group in the Rac or Meta as other literature reported [28]. This process generates radicals and may further combine into dimers which are easier oxidized. So, the low potential oxidation and reduction peak (0.60 V) may
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Figure 4. (A) The optimized structure of ractopamine and metaproterenol with their Mulliken charges. (B) The electrochemical oxidation.
Figure 5. (A) Cyclic voltammograms of 106 M ractopamine at AuNPs/GCE with different scan rates. (B) The peak current at 1.12 V of ractopamine vs. the scan rate.
Figure 6. (A) Cyclic voltammograms of 106 M metaproterenol at AuNPs/GCE with different scan rates. (B) The peak current at 1.12 V of metaproterenol vs. the scan rate.
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Figure 7. (A) Differential pulse voltammograms of different concentrations of ractopamine at AuNPs modified GCE. The pulse period: 0.2 s, amplitude: 50 mV. (B) The calibration curve between logarithm of concentration and the current of peak based on DPV.
Figure 8. (A) Differential pulse voltammograms of different concentrations of metaproterenol at AuNPs modified GCE. The pulse period: 0.2 s, amplitude: 50 mV. (B) The calibration curve between logarithm of concentration and the current of peak based on DPV.
be attributed to the electrochemical response of dimers which need more research. The CV curves of 106 M Rac at AuNPs/GCE under different scan rates are represented in Figure 5A. As shown, the oxidation peak current of Rac is increasing with the adding scan rates. It is found that the oxidation peak currents are proportional to the scan rates (R = 0.98), which indicates a typical surface-controlled electrochemical behavior of Rac (Figure 5B). Meanwhile, the same behavior happens to the Meta as illustrated in Figure 6.
3.3. The determination of Rac and Meta In order to detect Rac selectively, the DPVs are carried out as illustrated in Figure 7A. From Figure 7A, the oxidation peak currents of Rac are increasing with gradual addition of concentrations. A typical linear relationship with a correlation coefficient of 0.98 (Figure 7B) exists between peak currents and logarithm of concentration. The detection range is from 109 to 105 M and the limit of detection is 3 1010 M (S/N = 3). Similarly, the determination of Meta is shown in Figure 8 and the linear range is also from 109 to 105 M. Compared with other methods reported above, this proposed approach has advantages including time-saving (a few minutes), low cost, wide linear range and low detection limit.
4. Conclusion In this Letter, the electrochemical behaviors of Rac and Meta on the AuNPs modified electrode were studied by PM3 method and CV curves. The mechanism of electrochemical process of Rac and Meta was proposed and the surface-controlled electrochemical oxidation was studied. The AuNPs were applied in modification of GCE for detection of Rac and Meta. During the detection, AuNPs with good conductivity, larger specific surface area and negatively charged surface catalyzed the oxidation of Rac and Meta effectively. The linear range of Rac and Meta are from 109 to 105 M and the limit of detection is 3 1010 M (S/N = 3). The high selectivity and convenience of AuNPs modified GCE is promising for the detection of Rac and Meta in clinical applications. Furthermore, this simple sensing platform may be extended to the determination of other b-agonist analogs and bio-compounds with electroactive groups. Acknowledgements The authors gratefully acknowledge the financial support from the National Basic Research Program 973:2011CB932700, 2011CB932703, National Outstanding Youth Science Foundation under Contract No. 60825407, National Natural Science Fund Project under Contract No. 60877025, 61077044, 91123025, Beijing
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