Materials Science and Engineering C 55 (2015) 457–461
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Molecularly imprinted electrochemical sensor for the highly selective and sensitive determination of melamine Benzhi Liu ⁎, Bo Xiao, Liqiang Cui, Min Wang School of Environmental Science and Engineering, Yancheng Institute of Technology, Yancheng, Jiangsu Province, China
a r t i c l e
i n f o
Article history: Received 5 March 2015 Received in revised form 5 May 2015 Accepted 25 May 2015 Available online 31 May 2015 Keywords: Melamine Electrochemical sensor Molecularly imprinted polymer Milk products
a b s t r a c t A selective and sensitive molecularly imprinted electrochemical sensor was developed for the determination of melamine. Carbon nanotube–ionic liquid composite was used as a carrier to enhance the number of imprinted cavities and then improve the selectivity and sensitivity of the sensor. The proposed sensor showed a linear relationship with the concentration of melamine in the range of 0.4 to 9.2 μM, with a detection limit of 0.11 μM. The sensor was successfully applied to the determination of melamine in milk products with good recovery. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Melamine (1,3,5-triazine-2,4,6-triamine) is a kind of triazine analogue which has three amino groups. Because it contains a substantial amount of nitrogen (66% by mass), some unethical manufacturers added it to the milk products to obtain high protein contents. The US Food and Drug Administration (FDA), the European community, and other countries and regions have established criteria of maximum residue limits for melamine in various food products. Standard limits of 1 ppm for melamine in infant formula and 2.5 ppm in other milk products have been legislated by many countries [1]. Ingestion of melamine at levels above the safety limit can cause urinary system diseases, especially in babies and children [2]. Therefore, a sensitive and reliable method is essential for the determination of melamine in milk products for children. Many methods have been developed for the determination of melamine, such as liquid chromatography [3,4], capillary electrophoresis [5–7], chromatography– mass spectrometry [8–10], enzyme-linked immuno-assay [11] and electrochemical techniques [12–15]. Among these methods, electrochemical techniques have the characteristics of simplicity, low cost, accuracy and easy to on-site detection. Moreover, electrochemical techniques have provided a sensitive and selective approach for the detection of numerous biological and environmental compounds [16–24]. Thus, in recent years, electrochemical techniques have attracted more and more attentions in analytical chemistry due to their wide applications.
⁎ Corresponding author at: School of Environmental Science and Engineering, Yancheng Institute of Technology, Jianjun Road 211, Yancheng, Jiangsu Province, China. E-mail address: [email protected] (B. Liu).
http://dx.doi.org/10.1016/j.msec.2015.05.080 0928-4931/© 2015 Elsevier B.V. All rights reserved.
For the formation of specific recognition sites to improve the selectivity of electrochemical method, molecularly imprinting technique has been proposed and developed fastly in recent years. Firstly, the affinity matrix is prepared by the polymerization of functional monomers and crosslinkers in the presence of template molecules. Then, the template molecules are removed from the polymer. Thus, the molecularly imprinted polymer (MIP) is obtained with the specific cavities complementarily in shape, size and functional groups to the template molecule, which could selectively recognize template molecule. Molecularly imprinted electrochemical sensors have received considerable attention for the determination of melamine because of their high selectivity and stability [25–27]. Recently, functional nanomaterial is often used to improve the conductivity and catalytic ability of the sensors and then greatly enhance the intensity of the electrochemical signal [28]. Because of the large surface area, functional nanomaterial could also be used as a carrier in the preparation of molecularly imprinted polymers to increase the number of imprinted cavities. Carbon nanotubes (CNTs) have been widely used in electrochemical field due to their unique properties such as large specific surface area, good mechanical stability and high electronic conductivity [29–31]. Ionic liquid (IL) is a salt in the liquid state. IL is largely made of ions and short-lived ion pairs. Room temperature ionic liquids consist of bulky and asymmetric organic cations such as 1-alkyl-3-methylimidazolium, 1-alkylpyridinium, N-methyl-N-alkylpyrrolidinium and ammonium ions. It has unique properties such as good ionic conductivity, high viscosity, high chemical and thermal stabilities [32]. Thus, it has been widely used as powerful solvents and electrolytes in electrochemistry. The capability of IL combining with CNT to form conductive composite makes it very attractive for the synthesis of functional nanomaterial. Therefore, CNT–IL composite could be used as a kind of robust and advanced carrier material for the preparation of molecularly imprinted polymers.
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Pyrrole is an electroactive functional monomer, which is often employed to fabricate MIP sensor for a variety of molecules recognition and detection [33,34]. The binding sites of polypyrrole molecules could effectively bind to the melamine through hydrogen bonds. In this study, a molecularly imprinted electrochemical sensor was prepared for the determination of melamine. Pyrrole was used as monomer, CNT–IL composite was used as a carrier to enhance the number of imprinted cavities and then improve the selectivity and sensitivity of the sensor. The preparation process and the electrochemical response of the sensor to melamine were shown in Fig. 1. The proposed electrochemical sensor showed a high selectivity toward melamine as well as a broad linear range and a low detection limit under the optimized conditions. Satisfactory results were also obtained for the determination of melamine in real samples. 2. Experimental 2.1. Reagents and instrumentation Multi-walled carbon nanotubes (MWCNTs) were obtained from Shenzhen Nanotech Port Co., Ltd. (Shenzhen, China) with a typical diameter of 10–20 nm. The purity was more than 98%. The ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF4) was purchased from Lanzhou Centre for Green Chemistry and Catalysis, LICP, CAS (Lanzhou, China). All chemicals were of analytical grade and used as received without further purification. Melamine, glycine, and histidine were obtained from Tianjin Kermel Chemical Reagent Co., Ltd. (Tianjin, China). Pyrrole, lactose, and uric acid were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). All other chemical reagents were obtained from Nanjing Chemical Reagent Company (Nanjing China). All the solutions were prepared with double distilled water. All electrochemical experiments were carried out on a CS350 Electrochemical Workstation (Wuhan Corrtest Instruments CO., LTD, Wuhan, China). A conventional three-electrode cell configuration was employed for the electrochemical measurements. A modified glassy carbon electrode (disc diameter of 3 mm) was used as the working electrode, with saturated calomel electrode (SCE, saturated KCl) and platinum wire for the reference and the counter electrode, respectively. All the potentials are versus saturated calomel electrode (saturated KCl). Transmission electron microscopy (TEM) images were obtained by using JEM-200CX (JEOL Ltd., Japan).
substances. Subsequently, 10 μL of the composite dispersion was dropped on the surface of GCE and dried in air. The extraction of template molecules from polymer matrix is often using organic reagents or buffer solution as eluent. However, it is time consuming and the template cannot be removed entirely. In this work, a simple method, cyclic voltammetry was used to extract melamine molecules from the imprinted polymer matrix. The embedded melamine molecules were extracted by scanning the modified electrode between — 0.3 and 0.8 V in 0.3 mol L−1 KOH and 0.1 mol L−1 KCl for several cycles until no redox peak for melamine was observed, which demonstrated the complete removal of melamine. Thus, the CNT–IL/ MIP/GCE (where MIP is molecularly imprinted polymers) was obtained. Fabrication of CNT–IL/NIP/GCE (where NIP is non-molecularly imprinted polymers) was the same as the process mentioned above but without the addition of melamine. In addition, 10 μL of the CNT–IL composite dispersion was dropped on the clean GCE surface and then dried in air to prepare CNT–IL/GCE. The modified electrodes were stored in air at room temperature until used. 2.3. Experimental procedures A standard three-electrode cell connected to the CS350 Electrochemical Workstation was used for electrochemical measurements. The CNT–IL/MIP/GCE was immersed in the solution containing 0.2 mol L− 1 H2SO4, 0.1 mol L− 1 KCl and certain amount of melamine for 120 s, then the cyclic voltammograms were recorded from 0.2 to 0.9 V at a scan rate of 100 mV/s, the square wave voltammograms were recorded from 0.2 to 0.9 V with a step increment of 4 mV, amplitude of 25 mV and frequency of 15 Hz. 3. Results and discussion 3.1. Morphology characterization of CNT–IL/MIP composite To investigate the morphology of the fabricated CNT–IL/MIP composite, TEM images were carried out and results were shown in Fig. 2. The image of pure CNT shows a typically smooth surface in Fig. 2A. By contrast, the CNT–IL composite looks like gel-like substance (Fig. 2B). However, many particles were observed on the surface of CNT–IL composite after polymerization, which attributed to the formation of polypyrroles (Fig. 2C). 3.2. Electrochemical behavior of melamine at modified electrodes
2.2. Preparing CNT–IL/MIP composite and fabrication of modified electrode Prior to use, the glassy carbon electrode (GCE) was carefully polished with a leather containing 0.05 μm Al2 O 3 slurry and then ordinal ultrasonically cleaned in ethanol and distilled water. Firstly, 5 mg of the MWCNT was dispersed in 10 ml of [Bmim]BF4 by ultrasonic agitation for about 1 h. Then, the polymerization reaction was initiated with the addition of 0.3 mL H2O2 to the mixture containing 10 mL of CNT–IL composite, 0.1 mL of pyrrole, 0.02 g FeCl2, 0.1 g melamine and 40 mL water. After 3 h reaction with stirring, the reactants were washed with water to remove the unreacted
Fig. 3A shows the cyclic voltammograms (CVs) of 20 μM melamine at CNT–IL/MIP/GCE (a), CNT–IL/GCE (b) and CNT–IL/NIP/GCE (c) in the electrolyte solution containing 0.2 mol L−1 H2SO4 and 0.1 mol L−1 KCl recorded after 120 s preconcentration. As shown in curve a, a pair of well-defined redox peaks are observed, which is attributed to the redox reaction of melamine at CNT–IL/MIP/GCE. It is also found that melamine shows similar electrochemical response at CNT–IL/NIP/GCE or CNT–IL/GCE, however, the peak current is much lower than that of CNT–IL/MIP/GCE. The oxidation peak current of CNT–IL/MIP/GCE is about 3 times that of CNT–IL/NIP/GCE and 2.2 times that of CNT–IL/
Fig. 1. The preparation process of CNT–IL/MIP and CNT–IL/NIP, and the comparison of electrochemical response of melamine at different modified electrodes.
B. Liu et al. / Materials Science and Engineering C 55 (2015) 457–461
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Fig. 3. CVs of 20 μM melamine at CNT–IL/MIP/GCE (a), CNT–IL/GCE (b) and CNT–IL/NIP/ GCE (c) in the electrolyte solution (A), and effect of accumulation time on the current response (B).
Fig. 2. TEM images of CNT (A), CNT–IL (B) and CNT–IL/MIP (C).
GCE. The phenomenon could be explained that the CNT–IL matrix has a large surface area which could greatly increase the dispersion of large amount of MIP on the electrode surface. Consequently, the melamine concentration on the surface of electrode could be effectively increased due to the plenty of cavities situated in the MIP. These recognition cavities consistent to the molecular shape, size, and functionality of the template molecule, resulting in the specific binding ability of melamine. Therefore, the peak current of CNT–IL/MIP/GCE increased greatly comparing with other electrodes. The accumulation of melamine in electrolyte solution was investigated by varying the adsorption time from 0 to 240 s, and the initial concentration of melamine kept constant at 2.0 μM. As can be seen from Fig. 3B, the oxidation peak current increases rapidly with the adsorption time from 0 to 120 s and then levels off after 120 s. The result reveals rapid response equilibrium of melamine molecules to CNT–IL/MIP, which might be due to the surface binding sites of CNT–IL/MIP composite.
used for the determination of melamine. Fig. 4A shows the SWVs of CNT–IL/MIP/GCE in electrolyte solution containing different concentrations of melamine. As shown in Fig. 4B, the oxidation peak current of melamine linearly increase with the concentration of melamine in the range of 0.4 to 9.2 μM, with a detection limit of 0.11 μM. According to the IUPAC recommendation [35], detection limit is determined using 3σ/slope ratio, where σ is the standard deviation of the mean value for 10 determinations of the blank. The linear regression equation can be expressed as Ipa (μA) = 18.01 + 6.43c (μM), with a correlation coefficient r = 0.998. The comparison of the proposed sensor for melamine detection with other methods was shown in Table 1. It can be seen that the proposed sensor also has a wide linear range and low detection limit which make it suitable for the determination of trace melamine. 3.4. Repeatability, reproducibility and stability The repeatability was investigated for the determination of 2.0 μM melamine using the same sensor. The calculated RSD was about 3.9% (n = 5). This good repeatability reveals that the binding of melamine to the polymers is reversible and the sensor could be regenerated and used repeatedly. To investigate the reproducibility of the proposed sensor, a series of four sensors prepared in the same manner was tested and the RSD is 4.5%. When the prepared sensor is stored at room temperature after 10 days, the peak current response retains 92% of its initial response. These results indicated that the proposed sensor has good stability and reproducibility. 3.5. Selectivity study
3.3. Determination of melamine Square wave voltammetry (SWV) has a much higher current sensitivity and better resolution than cyclic voltammetry. Thus, SWV was
To verify the selectivity, glycine, histidine (His), ascorbic acid (AA), lactose, glucose, sucrose, and uric acid (UA) were selected in the interference experiments. The selective experiments were carried out by
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Fig. 4. (A) SWVs of CNT–IL/MIP/GCE in solution containing different concentrations of melamine, from 1–11: 0, 0.4, 1.2, 2.0, 3.6, 5.2, 6.8, 9.2, 10.2, 11.2, 12.4 μM, and (B) plot of peak current versus melamine concentration.
detecting the current response of 2.0 μM melamine at CNT–IL/MIP/GCE in the presence of 10-fold concentration of interference species. As can be seen in Fig. 5A, the above species did not show obvious interference to the melamine. For comparison, the electrochemical responses of interference species were studied. As shown in Fig. 5B, the current of interference species was close to the background current and much smaller than that of melamine. It is obvious that the species almost have no interference to the determination of melamine. In addition, the effect of several ions on the determination of melamine was also studied. The results showed that 300-fold concentration of Na+, K+, − Ca2+, Zn2+, Al3+, Mg2+, Cl−, SO2− 4 , and NO3 have no interference on the determination of melamine. The results suggested that the proposed sensor has good selectivity for the detection of melamine.
3.6. Real sample analysis To verify the application in real analysis, the CNT–IL/MIP/GCE was used to detect melamine in the liquid milk and infant formula which bought from a local supermarket. The liquid milk and infant formula
Table 1 The comparison of the proposed sensor for melamine detection with other methods. Materials and method
Linear range (μM)
LOD (μM)
References
Poly(para-aminobenzoic acid)/GCE Imprinted sol–gel electrochemical sensor Solid-phase extraction combined with MIP MIP potentiometric sensor CNT–IL/MIP
4.0–450 0.63–110 0.5–10.0 5.0–10,000 0.4–9.2
0.36 0.068 0.5 1.6 0.11
[14] [26] [36] [37] This work
Fig. 5. The peak current changes on CNT–IL/MIP/GCE with the addition of interference species (A) and the electrochemical responses of interference species (B).
were pretreated with the general procedure. Briefly, 5 mL milk (or 2 g infant formula) was first mixed with 5 ml of 0.5 mol L−1 trichloroacetic acid and 40 ml of methanol. After 10 min shaking and 20 min sonication, the mixture was centrifuged at 9000 rpm for 15 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was then condensed to give a total volume of 5 ml for detection. No melamine was detected in the real sample. Then the standard addition method was adopted to detect melamine in real samples. A certain amount of melamine was added to the same sample as purchased. Then, the sample was pretreated and detected in the same way. The results were shown in Table 2. It was found that the present method showed good recovery in the range of 93.3–96.7%, which demonstrated that the present sensor was reliable and effective for the determination of melamine in real samples.
Table 2 Analysis of melamine in real samples. Samples
Added (μM)
Found (μM)
Recovery (%)
Milk
0 1.2 3.6 0 1.2 3.6
0 1.12 3.41 0 1.16 3.45
– 93.3 94.7 – 96.7 95.8
Infant formula
RSD (%) (n = 3) 3.9 3.5 4.2 3.4
B. Liu et al. / Materials Science and Engineering C 55 (2015) 457–461
4. Conclusion In this work, we presented a simple, sensitive and selective molecularly imprinted electrochemical sensor for melamine detection. A linear relationship between melamine concentration and current response ranged from 0.4 to 9.2 μM with a low detection limit of 0.11 μM was obtained. The proposed sensor was applied for the detection of melamine in milk products with good recovery. The results indicated that the proposed electrochemical sensor might be potential to determine melamine in other real samples to ensure food safety. Acknowledgments This work was supported by the Talent Introduction Funds of Yancheng Institute of Technology (KJC2013003). References [1] J.S. Chen, A worldwide food safety concern in 2008 melamine-contaminated infant formula in China caused urinary tract stone in 290,000 children in China, Chin. Med. J. 122 (2009) 243–244. [2] N. Guan, Q.F. Fan, J. Ding, Y.M. Zhao, J.Q. Lu, Y. Ai, G.B. Xu, S.N. Zhu, C. Yao, L.N. Jiang, J. Miao, H. Zhang, D. Zhao, X.Y. Liu, Y.N. Yao, Melamine-contaminated powdered formula and urolithiasis in young children, N. Engl. J. Med. 360 (2009) 1067–1074. [3] S. Ehling, S. Tefera, I.P. Ho, High-performance liquid chromatographic method for the simultaneous detection of the adulteration of cereal flours with melamine and related triazine by-products ammeline, ammelide, and cyanuric acid, Food Addit. Contam. 24 (2007) 1319–1325. [4] M. Lin, L. He, J. Awika, L. Yang, D.R. Ledoux, H. Li, A. Mustapha, Detection of melamine in gluten, chicken feed, and processed foods using surface enhanced Raman spectroscopy and HPLC, J. Food Sci. 73 (2008) 129–134. [5] C.W. Klampfl, L. Andersen, M. Haunschmidt, M. Himmelsbach, W. Buch-berger, Analysis of melamine in milk powder by CZE using UV detection and hyphenation with ESI quadrupole/TOF MS detection, Electrophoresis 30 (2009) 1743–1746. [6] N. Yan, L. Zhou, Z. Zhu, X. Chen, Determination of melamine in dairy products, fish feed, and fish by capillary zone electrophoresis with diode array detection, J. Agric. Food Chem. 57 (2009) 807–811. [7] C. Zhai, W. Qiang, J. Sheng, J. Lei, H. Ju, Pretreatment-free fast ultraviolet detection of melamine in milk products with a disposable microfluidic device, J. Chromatogr. A 1217 (2010) 785–789. [8] W.C. Andersen, S.B. Turnipseed, C.M. Karbiwnyk, S.B. Clark, M.R. Madson, C.M. Gieseker, R.A. Miller, N.G. Rummel, R. Reimschuessel, Determination and confirmation of melamine residues in catfish, trout, tilapia, salmon, and shrimp by liquid chromatography with tandem mass spectrometry, J. Agric. Food Chem. 56 (2008) 4340–4347. [9] M.S. Filigenzi, E.R. Tor, R.H. Poppenga, L.A. Aston, B. Puschner, The determination of melamine in muscle tissue by liquid chromatography/tandem mass spectrometry, Rapid Commun. Mass Spectrom. 21 (2007) 4027–4032. [10] M.S. Filigenzi, B. Puschner, L.S. Aston, R.H. Poppenga, Diagnostic determination of melamine and related compounds in kidney tissue by liquid chromatography/tandem mass spectrometry, J. Agric. Food Chem. 56 (2008) 7593–7599. [11] E.A.E. Garber, Detection of melamine using commercial enzyme-linked immunosorbent assay technology, J. Food Prot. 71 (2008) 590–594. [12] Q. Cao, H. Zhao, Y. He, N. Ding, J. Wang, Electrochemical sensing of melamine with 3,4-dihydroxyphenylacetic acid as recognition element, Anal. Chim. Acta 675 (2010) 24–28. [13] C.W. Liao, Y.R. Chen, J.L. Chang, J.M. Zen, A sensitive electrochemical approach for melamine detection using a disposable screen printed carbon electrode, Electroanalysis 23 (2011) 573–576. [14] Y.T. Liu, J. Deng, X.L. Xiao, L. Ding, Y.L. Yuan, H. Li, X.T. Li, X.N. Yan, L.L. Wang, Electrochemical sensor based on a poly(para-aminobenzoic acid) film modified glassy carbon electrode for the determination of melamine in milk, Electrochim. Acta 56 (2011) 4595–4602. [15] H. Zhu, S. Zhang, M. Li, Y. Shao, Z. Zhu, Electrochemical sensor for melamine based on its copper complex, Chem. Commun. 46 (2010) 2259–2261. [16] H. Beitollahi, I. Sheikhshoaie, Selective voltammetric determination of norepinephrine in the presence of acetaminophen and folic acid at a modified carbon nanotube paste electrode, J. Electroanal. Chem. 661 (2011) 336–342.
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Molecularly imprinted electrochemical sensor for the highly selective and sensitive determination of melamine Benzhi Liu ⁎, Bo Xiao, Liqiang Cui, Min Wang School of Environmental Science and Engineering, Yancheng Institute of Technology, Yancheng, Jiangsu Province, China
a r t i c l e
i n f o
Article history: Received 5 March 2015 Received in revised form 5 May 2015 Accepted 25 May 2015 Available online 31 May 2015 Keywords: Melamine Electrochemical sensor Molecularly imprinted polymer Milk products
a b s t r a c t A selective and sensitive molecularly imprinted electrochemical sensor was developed for the determination of melamine. Carbon nanotube–ionic liquid composite was used as a carrier to enhance the number of imprinted cavities and then improve the selectivity and sensitivity of the sensor. The proposed sensor showed a linear relationship with the concentration of melamine in the range of 0.4 to 9.2 μM, with a detection limit of 0.11 μM. The sensor was successfully applied to the determination of melamine in milk products with good recovery. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Melamine (1,3,5-triazine-2,4,6-triamine) is a kind of triazine analogue which has three amino groups. Because it contains a substantial amount of nitrogen (66% by mass), some unethical manufacturers added it to the milk products to obtain high protein contents. The US Food and Drug Administration (FDA), the European community, and other countries and regions have established criteria of maximum residue limits for melamine in various food products. Standard limits of 1 ppm for melamine in infant formula and 2.5 ppm in other milk products have been legislated by many countries [1]. Ingestion of melamine at levels above the safety limit can cause urinary system diseases, especially in babies and children [2]. Therefore, a sensitive and reliable method is essential for the determination of melamine in milk products for children. Many methods have been developed for the determination of melamine, such as liquid chromatography [3,4], capillary electrophoresis [5–7], chromatography– mass spectrometry [8–10], enzyme-linked immuno-assay [11] and electrochemical techniques [12–15]. Among these methods, electrochemical techniques have the characteristics of simplicity, low cost, accuracy and easy to on-site detection. Moreover, electrochemical techniques have provided a sensitive and selective approach for the detection of numerous biological and environmental compounds [16–24]. Thus, in recent years, electrochemical techniques have attracted more and more attentions in analytical chemistry due to their wide applications.
⁎ Corresponding author at: School of Environmental Science and Engineering, Yancheng Institute of Technology, Jianjun Road 211, Yancheng, Jiangsu Province, China. E-mail address: [email protected] (B. Liu).
http://dx.doi.org/10.1016/j.msec.2015.05.080 0928-4931/© 2015 Elsevier B.V. All rights reserved.
For the formation of specific recognition sites to improve the selectivity of electrochemical method, molecularly imprinting technique has been proposed and developed fastly in recent years. Firstly, the affinity matrix is prepared by the polymerization of functional monomers and crosslinkers in the presence of template molecules. Then, the template molecules are removed from the polymer. Thus, the molecularly imprinted polymer (MIP) is obtained with the specific cavities complementarily in shape, size and functional groups to the template molecule, which could selectively recognize template molecule. Molecularly imprinted electrochemical sensors have received considerable attention for the determination of melamine because of their high selectivity and stability [25–27]. Recently, functional nanomaterial is often used to improve the conductivity and catalytic ability of the sensors and then greatly enhance the intensity of the electrochemical signal [28]. Because of the large surface area, functional nanomaterial could also be used as a carrier in the preparation of molecularly imprinted polymers to increase the number of imprinted cavities. Carbon nanotubes (CNTs) have been widely used in electrochemical field due to their unique properties such as large specific surface area, good mechanical stability and high electronic conductivity [29–31]. Ionic liquid (IL) is a salt in the liquid state. IL is largely made of ions and short-lived ion pairs. Room temperature ionic liquids consist of bulky and asymmetric organic cations such as 1-alkyl-3-methylimidazolium, 1-alkylpyridinium, N-methyl-N-alkylpyrrolidinium and ammonium ions. It has unique properties such as good ionic conductivity, high viscosity, high chemical and thermal stabilities [32]. Thus, it has been widely used as powerful solvents and electrolytes in electrochemistry. The capability of IL combining with CNT to form conductive composite makes it very attractive for the synthesis of functional nanomaterial. Therefore, CNT–IL composite could be used as a kind of robust and advanced carrier material for the preparation of molecularly imprinted polymers.
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Pyrrole is an electroactive functional monomer, which is often employed to fabricate MIP sensor for a variety of molecules recognition and detection [33,34]. The binding sites of polypyrrole molecules could effectively bind to the melamine through hydrogen bonds. In this study, a molecularly imprinted electrochemical sensor was prepared for the determination of melamine. Pyrrole was used as monomer, CNT–IL composite was used as a carrier to enhance the number of imprinted cavities and then improve the selectivity and sensitivity of the sensor. The preparation process and the electrochemical response of the sensor to melamine were shown in Fig. 1. The proposed electrochemical sensor showed a high selectivity toward melamine as well as a broad linear range and a low detection limit under the optimized conditions. Satisfactory results were also obtained for the determination of melamine in real samples. 2. Experimental 2.1. Reagents and instrumentation Multi-walled carbon nanotubes (MWCNTs) were obtained from Shenzhen Nanotech Port Co., Ltd. (Shenzhen, China) with a typical diameter of 10–20 nm. The purity was more than 98%. The ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF4) was purchased from Lanzhou Centre for Green Chemistry and Catalysis, LICP, CAS (Lanzhou, China). All chemicals were of analytical grade and used as received without further purification. Melamine, glycine, and histidine were obtained from Tianjin Kermel Chemical Reagent Co., Ltd. (Tianjin, China). Pyrrole, lactose, and uric acid were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). All other chemical reagents were obtained from Nanjing Chemical Reagent Company (Nanjing China). All the solutions were prepared with double distilled water. All electrochemical experiments were carried out on a CS350 Electrochemical Workstation (Wuhan Corrtest Instruments CO., LTD, Wuhan, China). A conventional three-electrode cell configuration was employed for the electrochemical measurements. A modified glassy carbon electrode (disc diameter of 3 mm) was used as the working electrode, with saturated calomel electrode (SCE, saturated KCl) and platinum wire for the reference and the counter electrode, respectively. All the potentials are versus saturated calomel electrode (saturated KCl). Transmission electron microscopy (TEM) images were obtained by using JEM-200CX (JEOL Ltd., Japan).
substances. Subsequently, 10 μL of the composite dispersion was dropped on the surface of GCE and dried in air. The extraction of template molecules from polymer matrix is often using organic reagents or buffer solution as eluent. However, it is time consuming and the template cannot be removed entirely. In this work, a simple method, cyclic voltammetry was used to extract melamine molecules from the imprinted polymer matrix. The embedded melamine molecules were extracted by scanning the modified electrode between — 0.3 and 0.8 V in 0.3 mol L−1 KOH and 0.1 mol L−1 KCl for several cycles until no redox peak for melamine was observed, which demonstrated the complete removal of melamine. Thus, the CNT–IL/ MIP/GCE (where MIP is molecularly imprinted polymers) was obtained. Fabrication of CNT–IL/NIP/GCE (where NIP is non-molecularly imprinted polymers) was the same as the process mentioned above but without the addition of melamine. In addition, 10 μL of the CNT–IL composite dispersion was dropped on the clean GCE surface and then dried in air to prepare CNT–IL/GCE. The modified electrodes were stored in air at room temperature until used. 2.3. Experimental procedures A standard three-electrode cell connected to the CS350 Electrochemical Workstation was used for electrochemical measurements. The CNT–IL/MIP/GCE was immersed in the solution containing 0.2 mol L− 1 H2SO4, 0.1 mol L− 1 KCl and certain amount of melamine for 120 s, then the cyclic voltammograms were recorded from 0.2 to 0.9 V at a scan rate of 100 mV/s, the square wave voltammograms were recorded from 0.2 to 0.9 V with a step increment of 4 mV, amplitude of 25 mV and frequency of 15 Hz. 3. Results and discussion 3.1. Morphology characterization of CNT–IL/MIP composite To investigate the morphology of the fabricated CNT–IL/MIP composite, TEM images were carried out and results were shown in Fig. 2. The image of pure CNT shows a typically smooth surface in Fig. 2A. By contrast, the CNT–IL composite looks like gel-like substance (Fig. 2B). However, many particles were observed on the surface of CNT–IL composite after polymerization, which attributed to the formation of polypyrroles (Fig. 2C). 3.2. Electrochemical behavior of melamine at modified electrodes
2.2. Preparing CNT–IL/MIP composite and fabrication of modified electrode Prior to use, the glassy carbon electrode (GCE) was carefully polished with a leather containing 0.05 μm Al2 O 3 slurry and then ordinal ultrasonically cleaned in ethanol and distilled water. Firstly, 5 mg of the MWCNT was dispersed in 10 ml of [Bmim]BF4 by ultrasonic agitation for about 1 h. Then, the polymerization reaction was initiated with the addition of 0.3 mL H2O2 to the mixture containing 10 mL of CNT–IL composite, 0.1 mL of pyrrole, 0.02 g FeCl2, 0.1 g melamine and 40 mL water. After 3 h reaction with stirring, the reactants were washed with water to remove the unreacted
Fig. 3A shows the cyclic voltammograms (CVs) of 20 μM melamine at CNT–IL/MIP/GCE (a), CNT–IL/GCE (b) and CNT–IL/NIP/GCE (c) in the electrolyte solution containing 0.2 mol L−1 H2SO4 and 0.1 mol L−1 KCl recorded after 120 s preconcentration. As shown in curve a, a pair of well-defined redox peaks are observed, which is attributed to the redox reaction of melamine at CNT–IL/MIP/GCE. It is also found that melamine shows similar electrochemical response at CNT–IL/NIP/GCE or CNT–IL/GCE, however, the peak current is much lower than that of CNT–IL/MIP/GCE. The oxidation peak current of CNT–IL/MIP/GCE is about 3 times that of CNT–IL/NIP/GCE and 2.2 times that of CNT–IL/
Fig. 1. The preparation process of CNT–IL/MIP and CNT–IL/NIP, and the comparison of electrochemical response of melamine at different modified electrodes.
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Fig. 3. CVs of 20 μM melamine at CNT–IL/MIP/GCE (a), CNT–IL/GCE (b) and CNT–IL/NIP/ GCE (c) in the electrolyte solution (A), and effect of accumulation time on the current response (B).
Fig. 2. TEM images of CNT (A), CNT–IL (B) and CNT–IL/MIP (C).
GCE. The phenomenon could be explained that the CNT–IL matrix has a large surface area which could greatly increase the dispersion of large amount of MIP on the electrode surface. Consequently, the melamine concentration on the surface of electrode could be effectively increased due to the plenty of cavities situated in the MIP. These recognition cavities consistent to the molecular shape, size, and functionality of the template molecule, resulting in the specific binding ability of melamine. Therefore, the peak current of CNT–IL/MIP/GCE increased greatly comparing with other electrodes. The accumulation of melamine in electrolyte solution was investigated by varying the adsorption time from 0 to 240 s, and the initial concentration of melamine kept constant at 2.0 μM. As can be seen from Fig. 3B, the oxidation peak current increases rapidly with the adsorption time from 0 to 120 s and then levels off after 120 s. The result reveals rapid response equilibrium of melamine molecules to CNT–IL/MIP, which might be due to the surface binding sites of CNT–IL/MIP composite.
used for the determination of melamine. Fig. 4A shows the SWVs of CNT–IL/MIP/GCE in electrolyte solution containing different concentrations of melamine. As shown in Fig. 4B, the oxidation peak current of melamine linearly increase with the concentration of melamine in the range of 0.4 to 9.2 μM, with a detection limit of 0.11 μM. According to the IUPAC recommendation [35], detection limit is determined using 3σ/slope ratio, where σ is the standard deviation of the mean value for 10 determinations of the blank. The linear regression equation can be expressed as Ipa (μA) = 18.01 + 6.43c (μM), with a correlation coefficient r = 0.998. The comparison of the proposed sensor for melamine detection with other methods was shown in Table 1. It can be seen that the proposed sensor also has a wide linear range and low detection limit which make it suitable for the determination of trace melamine. 3.4. Repeatability, reproducibility and stability The repeatability was investigated for the determination of 2.0 μM melamine using the same sensor. The calculated RSD was about 3.9% (n = 5). This good repeatability reveals that the binding of melamine to the polymers is reversible and the sensor could be regenerated and used repeatedly. To investigate the reproducibility of the proposed sensor, a series of four sensors prepared in the same manner was tested and the RSD is 4.5%. When the prepared sensor is stored at room temperature after 10 days, the peak current response retains 92% of its initial response. These results indicated that the proposed sensor has good stability and reproducibility. 3.5. Selectivity study
3.3. Determination of melamine Square wave voltammetry (SWV) has a much higher current sensitivity and better resolution than cyclic voltammetry. Thus, SWV was
To verify the selectivity, glycine, histidine (His), ascorbic acid (AA), lactose, glucose, sucrose, and uric acid (UA) were selected in the interference experiments. The selective experiments were carried out by
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Fig. 4. (A) SWVs of CNT–IL/MIP/GCE in solution containing different concentrations of melamine, from 1–11: 0, 0.4, 1.2, 2.0, 3.6, 5.2, 6.8, 9.2, 10.2, 11.2, 12.4 μM, and (B) plot of peak current versus melamine concentration.
detecting the current response of 2.0 μM melamine at CNT–IL/MIP/GCE in the presence of 10-fold concentration of interference species. As can be seen in Fig. 5A, the above species did not show obvious interference to the melamine. For comparison, the electrochemical responses of interference species were studied. As shown in Fig. 5B, the current of interference species was close to the background current and much smaller than that of melamine. It is obvious that the species almost have no interference to the determination of melamine. In addition, the effect of several ions on the determination of melamine was also studied. The results showed that 300-fold concentration of Na+, K+, − Ca2+, Zn2+, Al3+, Mg2+, Cl−, SO2− 4 , and NO3 have no interference on the determination of melamine. The results suggested that the proposed sensor has good selectivity for the detection of melamine.
3.6. Real sample analysis To verify the application in real analysis, the CNT–IL/MIP/GCE was used to detect melamine in the liquid milk and infant formula which bought from a local supermarket. The liquid milk and infant formula
Table 1 The comparison of the proposed sensor for melamine detection with other methods. Materials and method
Linear range (μM)
LOD (μM)
References
Poly(para-aminobenzoic acid)/GCE Imprinted sol–gel electrochemical sensor Solid-phase extraction combined with MIP MIP potentiometric sensor CNT–IL/MIP
4.0–450 0.63–110 0.5–10.0 5.0–10,000 0.4–9.2
0.36 0.068 0.5 1.6 0.11
[14] [26] [36] [37] This work
Fig. 5. The peak current changes on CNT–IL/MIP/GCE with the addition of interference species (A) and the electrochemical responses of interference species (B).
were pretreated with the general procedure. Briefly, 5 mL milk (or 2 g infant formula) was first mixed with 5 ml of 0.5 mol L−1 trichloroacetic acid and 40 ml of methanol. After 10 min shaking and 20 min sonication, the mixture was centrifuged at 9000 rpm for 15 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was then condensed to give a total volume of 5 ml for detection. No melamine was detected in the real sample. Then the standard addition method was adopted to detect melamine in real samples. A certain amount of melamine was added to the same sample as purchased. Then, the sample was pretreated and detected in the same way. The results were shown in Table 2. It was found that the present method showed good recovery in the range of 93.3–96.7%, which demonstrated that the present sensor was reliable and effective for the determination of melamine in real samples.
Table 2 Analysis of melamine in real samples. Samples
Added (μM)
Found (μM)
Recovery (%)
Milk
0 1.2 3.6 0 1.2 3.6
0 1.12 3.41 0 1.16 3.45
– 93.3 94.7 – 96.7 95.8
Infant formula
RSD (%) (n = 3) 3.9 3.5 4.2 3.4
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