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Development and characterization of an amperometric sensor for triclosan detection based on electropolymerized molecularly imprinted polymer
Microchemical Journal 91 (2009) 222–226
Contents lists available at ScienceDirect
Microchemical Journal j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i c r o c
Development and characterization of an amperometric sensor for triclosan detection based on electropolymerized molecularly imprinted polymer Ying Liu, Qi-Jun Song ⁎, Li Wang School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, Jiangsu, PR China
a r t i c l e
i n f o
Article history: Received 1 October 2008 Received in revised form 27 November 2008 Accepted 27 November 2008 Available online 6 December 2008 Keywords: O-phenylenediamine Molecularly imprinted polymer Electropolymerization Triclosan
a b s t r a c t A novel amperometric sensor based on electropolymerized molecularly imprinted polymer (MIP) for triclosan detection is reported. The sensor was prepared by electropolymerizing o-phenylenediamine (o-PD) on a glassy carbon electrode in the presence of template triclosan. The template can be quickly removed by NaOH solution. After incubating in acetate buffer for 15 min, the sensor response sensitively to triclosan over a linear range of 2.0 × 10− 7 to 3.0 × 10− 6 mol/L and a detection limit as low as 8.0 × 10− 8 mol/L is obtained. This sensor provides an efficient way for eliminating interferences from compounds with similar structures to that of triclosan. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Triclosan (2,2,4′-trichloro-2′-hydroxydiphenyl ether, Fig. 1a) is an antimicrobial compound. Due to its excellent performances triclosan has been incorporated into over 200 light industrial products, including oral sanitary products, cosmetics, liquid detergent, medical disinfectant, anti-bacterial fabric softener and anti-bacterial polymeric substance [1]. As a consumer product ingredient, triclosan enters sewer systems and is transported to wastewater sewage treatment plants. Triclosan has therefore been widely found in rivers, lakes and open sea water at ng/L levels [2–4]. Recently attentions have been paid to triclosan and its degradation products partly due to their close relation in chemical structure with that of the highly toxic contaminants – dioxins. Triclosan can undergo cyclization to form 2,8-dichlorodibenzo-p-dioxin (2,8-DCDD, Fig. 1b) in aqueous solution under UV irradiation [5–6]. Furthermore, 2, 4dichlorophenol and 2, 4, 6-trichlorophenol have been detected as the degradation products of triclosan in water or in chlorinated waters [7–8]. Therefore, it is necessary to develop sensitive and selective analytical method for triclosan in order to support investigations of its environmental fate. Instrumental methods for triclosan determination are developed, including gas chromatography–atomic emission detection(GC-AED) [9], gas chromatography-ion trap mass spectrometry (GC-ITMS)[10], liquid chromatography–ultraviolet detector(LC-UVD)[11], liquid chromatography–mass spectroscopy (LC-MS) [12] and electrochemical methods [13– 19]. Recently our group have also developed a chemiluminescence (CL) ⁎ Corresponding author. E-mail address: [email protected] (Q.-J. Song). 0026-265X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2008.11.007
method for triclosan determination[20]. Although GC and LC are wellestablished methods with a low detection limit, the bulky and expensive apparatus still hinder their practical applications. Electrochemical and CL methods for triclosan determinations are fast, convenient and with less expensive instruments, but strongly adsorbed oligomeric products produced by the oxidation of triclosan [13] on electrode surface may interfere with the electrochemical detection, while for CL method, pretransformation is needed and the selectivity is not good enough for direct application in water analysis. Therefore there still remains a great need for a fast and user-friendly in situ monitoring device of triclosan detection. This paper reports a new strategy by constructing molecularly imprinted polymers (MIPs) sensors for triclosan determination. MIPs sensors are proven to have advantages such as excellent selectivity and stability [21–23]. MIPs have been developed for a large variety of molecules including amino acids, mycotoxins, nucleotide bases, pesticides, pharmaceuticals, proteins and vitamins in last decades. Incorporation of the MIPs with electrochemical detection is therefore considered to be a promising method for triclosan determination. An amperometric sensor based on triclosan-imprinted poly-o-phenylenediamine (PPD) are fabricated in this work. O-phenylenediamine was chosen as monomer of electropolymerization, because it was proved to be easily electropolymerized on various substrate materials and form films with good chemical and mechanical stability [24–26]. Meanwhile, H-bonds can be formed between o-phenylenediamine and triclosan in acid medium, thus triclosan would bind with o-PD during electropolymerizing process and leave cavities matching triclosan after the removal of the template. Considering that PPD films and triclosan are electro-inactive in acid medium in the potential window chosen, an electroactive substance, ferrocyanide, was chosen as the mediator between the
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Fig. 1. Chemical structures of triclosan (a) and 2,8-dichlorodibenzo-p-dioxin (2,8-DCDD) (b).
imprinted electrodes and substrate solutions. A relationship between the current signal produced by ferrocyanide and the concentration of triclosan in supporting electrolytes can be obtained. 2. Experimental 2.1. Materials Triclosan (purity N 99.5%) was purchased from Alfa Aesar. 2,8dichlorodibenzo-p-dioxin (2,8-DCDD) (purity N 98%) was purchased from AccuStandard., USA. Other chemicals were obtained from Sinopharm Chemical Reagent Company (Shanghai, China). They are at least of analytical grade and used without further purification. Ultra pure water (18 MΩ) was used throughout the experiment. 2.2. Instruments Electrochemical measurements were performed on an IM6 electrochemical workstation (Zahner, Germany). A three-electrode configuration was used with a bare or polymer-coated glassy carbon (GC) electrode (4 mm in diameter) as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum wire(2 mm in diameter and 6 mm in length) as the auxiliary electrode. Prior to the experiments, GC electrode was subsequentially polished on a microcloth(chamois leather) with 0.5 and 0.05 μm aqueous slurry of alumina, and then it was alternately washed with water and alcohol. 2.3. Preparation of imprinted films Polymerization of o-PD was conducted by cyclic voltammetry (20 scans) in the range 0–0.8 V (scan rate 50 mV/s) in acetate buffer (pH 5.2) containing 6 mmol/L o-PD. For imprinted polymerization, triclosan was also added at a concentration 0.1 mmol/L. After the electropolymerization, the triclosan-imprinted PPD (iPPD) films were prepared by dipping the electrodes in 0.1 mol/L NaOH solution for 10 min to remove the template. Blank electrodes,
Fig. 2. Cyclic voltammograms for the electropolymerization of 6 mol/L o-PD containing 0.1 mmol/L triclosan at a GC electrode in acetate buffer solution (pH 5.2). Scan rate: 50 mV/s, sweep cycle: 20.
Fig. 3. Cyclic voltammograms of PPD film in borax buffer (pH 9.2) in the presence of triclosan (a) and in the absence of triclosan (b). Scan rate: 100 mV/s.
with non-imprinted PPD film modification, were prepared under the same conditions but in the absence of triclosan. 2.4. Verification of the imprinting effect The interaction between triclosan and imprinted PPD was evaluated by incubating the iPPD electrode in acetate buffer (pH 5.2) containing appropriate concentration of triclosan for 15 min under stirring. Then the films were dipped in acetate buffer for 3 min and washed by water for 15 s to remove the possible adsorptive substances on the surface. Electrochemical properties of the films were measured by cyclic voltammetry in 0.01 mol/L K4[Fe(CN)6] solution containing 1 mol/L KNO3 as the supporting electrolyte. 3. Results and discussion 3.1. Molecular imprinting electropolymerization The typical cyclic voltammograms recorded during the electropolymerization of o-PD on GC electrode in the presence of triclosan are shown in Fig. 2. The peak current drops dramatically with each scan. The oxidation wave appears completely irreversible. Finally the current approaches to zero, indicating the formation of nonconducting film, which hinders the monomer access to electrode surface. No significant difference was observed between the cyclic voltammograms obtained in the presence of triclosan and in its absence, which can be explained by the fact that triclosan does not have any electroactivity on GC electrode in the potential window
Fig. 4. Cyclic voltammograms for three different electrodes in 0.01 mol/L K4[Fe(CN)6] solutions. (a) Bare GC electrode; (b) GC electrode modified with iPPD film; (c) GC electrode modified with non-imprinted PPD film. Scan rate: 100 mV/s.
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Fig. 5. Removing time curves of template. The soaking time was: (a) 0; (b) 5; (c) 10; (d) 15; (e) 30 min. Scan rate: 100 mV/s.
chosen for the polymerization in acetate buffer (pH 5.2) [14]. As a result, the structure of triclosan is not electrochemically changed in the polymerization process. 3.2. Electrochemical characterization of MIP films In order to confirm whether triclosan has been embedded in PPD film, cyclic voltammograms of the PPD film prepared in the presence or absence of triclosan were respectively recorded in borax buffer (pH 9.2). Fig. 3 shows a typical comparison of cyclic voltammograms between the two different electrodes. In the absence of triclosan (Fig. 3b) no oxidation peak can be observed. However, in the presence of triclosan (Fig. 3a) an oxidation peak with a plateau shape can be clearly observed, which corresponds to the oxidation of triclosan and means that triclosan has been embedded in PPD film. Compared to the cyclic voltammograms of free triclosan in free in solution on bare electrode[13], oxidation peak of triclosan embedded in PPD film is more wide in shape and more positive in potential. This may be explained by the interaction between triclosan and the iPPD film, which make the electron transfer of triclosan embedded in film more difficult than triclosan free in solution. To further characterize the prepared electrodes, cyclic voltammograms of bare GC electrode, non-imprinted PPD electrode and iPPD electrode were performed in 0.01 mol/L K4[Fe(CN)6] solutions containing 1 mol/L KNO3. Fig. 4 shows a typical comparison of cyclic voltammograms among the three types of GC electrodes. The cyclic voltammetric results indicated that quasi-reversible electrochemical redox reactions of [Fe(CN)6]3−/[Fe(CN)6] 4− ion pair occurred with the bare GC electrode (Fig. 4a) with the peak potential differences of 130 mV
Fig. 6. Incubation time curves of the imprinted sensor. Incubation time was: (a) 0; (b) 5; (c) 10; (d) 15; (e) 20 min. Scan rate: 100 mV/s.
Fig. 7. A. Cyclic voltammograms of triclosan of different concentration in 0.01 mol/L K4 [Fe(CN)6] solutions containing 1 mol/L KNO3. (a) 0; (b) 8.0 × 10− 7; (c) 1.6 × 10− 6; (d) 2.4 × 10− 6; (e) 3.2 × 10− 6; (f) 4.0 × 10− 6; (g) 4.8 × 10− 6; (h) 5.6 × 10−5 mol/L. Scan rate: 100 mV/s.
and the ratio of the peak currents of about 1;1 respectively. As expected, almost no electrochemistry properties could be observed with the nonimprinted PPD electrode (Fig. 4c), which indicates that electro-inactive PPD film completely covered on the surface of the GC electrode and hindered ferrocyanide access to electrode surface. It is also noted that comparing to the bare electrode, an obvious current decrease appeared by using the electrode with iPPD film (Fig. 3b). This behavior is attributed to the presence of cavities that enable ferrocyanide diffuse through iPPD polymer towards the GC electrode, in contrast with non-imprinted PPD electrode, no redox peak can be observed. 3.3. Analytical features Triclosan can be determined by direct or indirect method using the iPPD film sensor. Direct method is based on the properties of triclosan easily producing oxidation peak through cavities existing in iPPD film in borax buffer. The determination of the oxidation peak current of triclosan is simple in operation, but the oxidation of triclosan at electrode surface is usually associated with the formation of an insulating polymeric blocking film[13]. Our experiments also found that the oxidation peak current of triclosan in iPPD electrode gradually decreases with the increase of scan times in borax buffer, which make the continuous measurement by direct method infeasible. In the indirect method, ferrocyanide is used as the mediator between the imprinted electrodes and substrate solutions containing triclosan. When the iPPD electrode was incubated in solution containing triclosan, the cavities in film were partially occupied by triclosan, which would lead to the
Fig. 8. Relative current changes in the iPPD sensor for different concentrations of triclosan and some interferents. (a) triclosan; (b) catechol; (c) 2,4,6-trichlorophenol; (d) 2,4-dichlorophenol; (e) 2,8-DCDD; (f) 1-(2-Pyridylazo)-2-naphthol.
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decrease of current signal produced by ferrocyanide. The higher the concentration of triclosan is, the lower the current would be. As is known, ferrocyanide possesses good electrochemical stability and can assure more reliable analytical results than the direct method. Indirect method was therefore preferred for the determination of triclosan. 3.3.1. Template removing treatment It is difficult to achieve a satisfactory sensitivity and reproducibility if template molecule cannot be eluted completely. Alcohol, ultra pure water and NaOH solution were respectively applied to remove template. The results indicated that alcohol and water can only partially remove the template, while NaOH solution can remove it more quickly and completely because triclosan can easily dissolve in NaOH solution. Cyclic voltammograms in 0.01 mol/L K4[Fe(CN)6] solution were performed after the iPPD electrode was dipped in NaOH solution for different time intervals. Fig. 5 shows the voltammograms corresponding to different soaking times. As can be seen, the current increased rapidly with the increase of soaking time at the beginning, but gradually approached stable after the soaking time was more than 10 min. So soaking in 0.1 mol/L NaOH solution for 10 min was chosen as the best condition for template removal. 3.3.2. Incubation time Incubation involved in the measurements is another critical factor for the performance of imprinted sensor. After template was removed from film, the iPPD sensor was incubated in acetate buffer solution (pH 5.2) containing triclosan. Fig. 6 shows the results obtained with different incubation times. The reduction peak current decreases with increasing incubation time, and a stable response is obtained after 15 min incubation. Thus, the optimum incubation time should be 15 min for the determination of triclosan. 3.3.3. The dependence of the current on the concentration of triclosan Cyclic voltammograms in 0.01 mol/L K4[Fe(CN)6] solution were performed after the iPPD electrode was incubated in solutions containing triclosan of different concentration for 15 min. Fig. 7A shows the dependence of the reduction current on the concentration of triclosan. As can be seen, the peak current decreases with the increase of triclosan concentration, due to the increasing number of the binding sites in the film were occupied by triclosan molecules. A calibration curve between the relative reduction peak current and the triclosan concentration is exhibited in Fig. 7B. Considering the absolute currents may vary with surface areas of the electrodes used and the distant between work electrode and auxiliary electrode in each experiment, relative current change (Δi/i0) was therefore suggested to indicate current response. Here, Δi=i0 −ic, i0 and ic is the current when concentration of triclosan is 0 and c mol/L respectively. As can be seen, at the high concentration range, the relative current change tend to be stable, which indicated that the imprinting sites were gradually occupied by triclosan molecules. The appearance of a platform in the curve reveals that the imprint cavities are saturated. The linear range is therefore estimated to be 2.0×10− 7 to 3.0×10− 6 mol/L and the detection limit is calculated to be 8.0×10− 8 mol/L based on the 3σ of blank signals. The linear calibration graphs of (Δi/i0) versus concentration of triclosan (c) can be described by the following equation, y=Δi/i0 =0.1079 c (µmol/L)+0.0078, and the correlation coefficient is 0.9968. 3.3.4. Selectivity of the sensor MIPs have specific selectivity towards the template molecule [27]. The specific recognition is based on the interaction between the template and the imprinting sites. To verify the specificity of iPPD sensor towards triclosan, interference experiments were performed. Compounds including catechol, 1-(2-Pyridylazo)-2-naphthol, 2,4dichlorophenol, 2,4,6-trichlorophenol and 2,8-DCDD were added into the supporting electrolyte respectively. As shown in Fig. 8, almost no interference was observed with 1-(2-Pyridylazo)-2-naphthol, which is
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larger in size than triclosan and cannot enter into the imprinting sites present in iPPD film. Only a slight interference was obtained with 2,8DCDD, which is similar to triclosan in structure, but still mismatch the imprinting sites and shows good selectivity. Comparatively, interferences produced by catechol, 2,4-dichlorophenol and 2,4,6-trichlorophenol are slightly larger than other substances, which may be explained by the fact that these compounds are more small than triclosan in molecular size and have some chances of approaching the imprinting sites. In view of above facts, the recognition sites formed in the polymerized film have the capability to distinguish target molecules through their size, shape and functional group distribution. 4. Conclusion We have combined the use of MIPs and electropolymerization to construct an electrochemical sensor that is capable of indirectly detecting triclosan. Compared to conventional direct electrochemical determination, this method can avoid the foul of easily absorbed oligomeric products formed by triclosan's oxidation on electrode surface. Optimum analytical conditions have been established, and the constructed sensor has demonstrated to have good selectivity to triclosan determination. The strategy proposed in this work is potentially applicable to the electrochemical determination of other important molecules without electrochemical activities. Acknowledgement We are grateful to acknowledge the financial support of Jiangnan University Youth Fund in China (No. 20070344). References [1] H. Singer, S. Müller, C. Tixier, L. Pillonel, Triclosan: occurrence and fate of a widely used biocide in the aquatic environment: field measurements in wastewater treatment plants, surface waters, and lake sediments, Environ. Sci. Technol. 36 (2002) 4998–5004. [2] D.W. Kolpin, M. Skopec, M.T. Meyer, E.T. Furlong, S.D. Zaugg, Urban contribution of pharmaceuticals and other organic wastewater contaminants to streams during differing flow conditions, Sci. Total Environ. 328 (2004) 119–130. [3] P.M. Thomas, G.D. Foster, Determination of nonsteroidal anti-inflammatory drugs, caffeine, and triclosan in wastewater by gas chromatography-mass spectrometry, J. Environ. Sci. Health, A 39 (2004) 1969–1978. [4] D.W. Kolpin, E.T. Furlong, M.T. Meyer, E.M. Thurman, S.D. Zaugg, L.B. Barber, H.T. Buxton, Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999–2000: A national reconnaissance, Environ. Sci. Technol. 36 (2002) 1202–1211. [5] D.E. Latch, J.L. Packer, W.A. Arnold, K. McNeil, Photochemical conversion of triclosan to 2,8-dichlorodibenzo-p-dioxin in aqueous solution, J. Photochem. Photobiol., A 158 (2003) 63–66. [6] M. Mezcua, M.J. Gómez, I. Ferrer, A. Aguera, M.D. Hernando, A.R. Fernández-Alba, Evidence of 2,7/2,8-dibenzodichloro-p-dioxin as a photodegradation product of triclosan in water and wastewater samples, Anal. Chim. Acta 524 (2004) 241–247. [7] P. Canosa, S. Morales, I. Rodriguez, E. Rubi, R. Cela, M. Gomez, Aquatic degradation of triclosan and formation of toxic chlorophenols in presence of low concentrations of free chlorine, Anal. Bioanal. Chem. 383 (2005) 1119–1126. [8] A.E. Greyshock, P.J. Vikesland, Environ, triclosan reactivity in chloraminated waters, Sci. Technol. 40 (2006) 2615–2622. [9] H.T. Rasmussen, R. McDonough, R.J. Gargiullo, B.P. McPherson, Determination of triclosan in human dental plaque by gas chromatography with atomic emission detection, J. High Resolut. Chromatogr. 19 (1996) 359–361. [10] J.L. Wu, N.P. Lama, D. Martens, A. Kettrup, Z.W. Cai, Triclosan determination in water related to wastewater treatment, Talanta 72 (2007) 1650–1654. [11] A. Sanches-Silva, R. Sendón-García, J. López-Hernández, P. Paseiro-Losada, Determination of triclosan in foodstuffs, J. Sep. Sci. 28 (2005) 65–72. [12] S.G. Chu, C.D. Metcalfe, Simultaneous determination of triclocarban and triclosan in municipal biosolids by liquid chromatography tandem mass spectrometry, J. Chromatogr., A 1164 (2007) 212–218. [13] M. Amiri, S. Shahrokhian, E. Psillakis, F. Marken, Electrostatic accumulation and determination of triclosan in ultrathin carbon nanoparticle composite film electrodes, Anal. Chim. Acta 593 (2007) 117–122. [14] R.M. Pemberton, J.P. Hart, Electrochemical behaviour of triclosan at a screenprinted carbon electrode and its voltammetric determination in toothpaste and mouthrinse products, Anal. Chim. Acta 390 (1999) 107–115. [15] P. Raghupathy, J. Mathiyarasu, James Joseph, K.L.N. Phani, V. Yegnaraman, Hydrotrope-driven disruption of micellar encapsulants for voltammetric detection of triclosan, J. Electroanal. Chem. 584 (2005) 210–214.
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[16] L. Vidal, A. Chisvert, A. Canals, E. Psillakis, A. Lapkin, F. Acosta, K.J. Edler, J.A. Holdaway, F. Marken, Chemically surface-modified carbon nanoparticle carrier for phenolic pollutants: extraction and electrochemical determination of benzophenone-3 and triclosan, Anal. Chim. Acta 616 (2008) 28–35. [17] L.H. Wang, S.C. Chu, C.Y. Chin, An electrochemical study of the reduction of triclosan (Irgasan DP-300) on carbon fiber ultramicroelectrodes, Bull. Electrochem. 20 (2004) 225–229. [18] M.J. Bonne, K.J. Edler, J.G. Buchanan, D. Wolverson, E. Psillakis, M. Helton, W. Thielemans, F. Marken, Thin-film modified electrodes with reconstituted cellulose-PDDAC films for the accumulation and detection of triclosan, J. Phys. Chem., C 112 (2008) 2660–2666. [19] A. Safavi, N. Maleki, H.R. Shahbaazi, Electrochemical determination of triclosan at a mercury electrode, Anal. Chim. Acta 494 (2003) 225–233. [20] S.J. Song, Q.J. Song, Z.L. Chen, Online phototransformation-flow injection chemiluminescence determination of triclosan, Anal. Bioanal. Chem. 387 (2007) 2917–2922. [21] H. Shiigi, H. Yakabe, M. Kishimoto, D. Kijima, Y. Zhang, U. Sree, B.A. Deore, T. Nagaoka, Molecularly imprinted overoxidized polypyrrole colloids: promising materials for molecular recognition, Microchim. Acta 143 (2003) 155–162.
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Contents lists available at ScienceDirect
Microchemical Journal j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i c r o c
Development and characterization of an amperometric sensor for triclosan detection based on electropolymerized molecularly imprinted polymer Ying Liu, Qi-Jun Song ⁎, Li Wang School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, Jiangsu, PR China
a r t i c l e
i n f o
Article history: Received 1 October 2008 Received in revised form 27 November 2008 Accepted 27 November 2008 Available online 6 December 2008 Keywords: O-phenylenediamine Molecularly imprinted polymer Electropolymerization Triclosan
a b s t r a c t A novel amperometric sensor based on electropolymerized molecularly imprinted polymer (MIP) for triclosan detection is reported. The sensor was prepared by electropolymerizing o-phenylenediamine (o-PD) on a glassy carbon electrode in the presence of template triclosan. The template can be quickly removed by NaOH solution. After incubating in acetate buffer for 15 min, the sensor response sensitively to triclosan over a linear range of 2.0 × 10− 7 to 3.0 × 10− 6 mol/L and a detection limit as low as 8.0 × 10− 8 mol/L is obtained. This sensor provides an efficient way for eliminating interferences from compounds with similar structures to that of triclosan. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Triclosan (2,2,4′-trichloro-2′-hydroxydiphenyl ether, Fig. 1a) is an antimicrobial compound. Due to its excellent performances triclosan has been incorporated into over 200 light industrial products, including oral sanitary products, cosmetics, liquid detergent, medical disinfectant, anti-bacterial fabric softener and anti-bacterial polymeric substance [1]. As a consumer product ingredient, triclosan enters sewer systems and is transported to wastewater sewage treatment plants. Triclosan has therefore been widely found in rivers, lakes and open sea water at ng/L levels [2–4]. Recently attentions have been paid to triclosan and its degradation products partly due to their close relation in chemical structure with that of the highly toxic contaminants – dioxins. Triclosan can undergo cyclization to form 2,8-dichlorodibenzo-p-dioxin (2,8-DCDD, Fig. 1b) in aqueous solution under UV irradiation [5–6]. Furthermore, 2, 4dichlorophenol and 2, 4, 6-trichlorophenol have been detected as the degradation products of triclosan in water or in chlorinated waters [7–8]. Therefore, it is necessary to develop sensitive and selective analytical method for triclosan in order to support investigations of its environmental fate. Instrumental methods for triclosan determination are developed, including gas chromatography–atomic emission detection(GC-AED) [9], gas chromatography-ion trap mass spectrometry (GC-ITMS)[10], liquid chromatography–ultraviolet detector(LC-UVD)[11], liquid chromatography–mass spectroscopy (LC-MS) [12] and electrochemical methods [13– 19]. Recently our group have also developed a chemiluminescence (CL) ⁎ Corresponding author. E-mail address: [email protected] (Q.-J. Song). 0026-265X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2008.11.007
method for triclosan determination[20]. Although GC and LC are wellestablished methods with a low detection limit, the bulky and expensive apparatus still hinder their practical applications. Electrochemical and CL methods for triclosan determinations are fast, convenient and with less expensive instruments, but strongly adsorbed oligomeric products produced by the oxidation of triclosan [13] on electrode surface may interfere with the electrochemical detection, while for CL method, pretransformation is needed and the selectivity is not good enough for direct application in water analysis. Therefore there still remains a great need for a fast and user-friendly in situ monitoring device of triclosan detection. This paper reports a new strategy by constructing molecularly imprinted polymers (MIPs) sensors for triclosan determination. MIPs sensors are proven to have advantages such as excellent selectivity and stability [21–23]. MIPs have been developed for a large variety of molecules including amino acids, mycotoxins, nucleotide bases, pesticides, pharmaceuticals, proteins and vitamins in last decades. Incorporation of the MIPs with electrochemical detection is therefore considered to be a promising method for triclosan determination. An amperometric sensor based on triclosan-imprinted poly-o-phenylenediamine (PPD) are fabricated in this work. O-phenylenediamine was chosen as monomer of electropolymerization, because it was proved to be easily electropolymerized on various substrate materials and form films with good chemical and mechanical stability [24–26]. Meanwhile, H-bonds can be formed between o-phenylenediamine and triclosan in acid medium, thus triclosan would bind with o-PD during electropolymerizing process and leave cavities matching triclosan after the removal of the template. Considering that PPD films and triclosan are electro-inactive in acid medium in the potential window chosen, an electroactive substance, ferrocyanide, was chosen as the mediator between the
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Fig. 1. Chemical structures of triclosan (a) and 2,8-dichlorodibenzo-p-dioxin (2,8-DCDD) (b).
imprinted electrodes and substrate solutions. A relationship between the current signal produced by ferrocyanide and the concentration of triclosan in supporting electrolytes can be obtained. 2. Experimental 2.1. Materials Triclosan (purity N 99.5%) was purchased from Alfa Aesar. 2,8dichlorodibenzo-p-dioxin (2,8-DCDD) (purity N 98%) was purchased from AccuStandard., USA. Other chemicals were obtained from Sinopharm Chemical Reagent Company (Shanghai, China). They are at least of analytical grade and used without further purification. Ultra pure water (18 MΩ) was used throughout the experiment. 2.2. Instruments Electrochemical measurements were performed on an IM6 electrochemical workstation (Zahner, Germany). A three-electrode configuration was used with a bare or polymer-coated glassy carbon (GC) electrode (4 mm in diameter) as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum wire(2 mm in diameter and 6 mm in length) as the auxiliary electrode. Prior to the experiments, GC electrode was subsequentially polished on a microcloth(chamois leather) with 0.5 and 0.05 μm aqueous slurry of alumina, and then it was alternately washed with water and alcohol. 2.3. Preparation of imprinted films Polymerization of o-PD was conducted by cyclic voltammetry (20 scans) in the range 0–0.8 V (scan rate 50 mV/s) in acetate buffer (pH 5.2) containing 6 mmol/L o-PD. For imprinted polymerization, triclosan was also added at a concentration 0.1 mmol/L. After the electropolymerization, the triclosan-imprinted PPD (iPPD) films were prepared by dipping the electrodes in 0.1 mol/L NaOH solution for 10 min to remove the template. Blank electrodes,
Fig. 2. Cyclic voltammograms for the electropolymerization of 6 mol/L o-PD containing 0.1 mmol/L triclosan at a GC electrode in acetate buffer solution (pH 5.2). Scan rate: 50 mV/s, sweep cycle: 20.
Fig. 3. Cyclic voltammograms of PPD film in borax buffer (pH 9.2) in the presence of triclosan (a) and in the absence of triclosan (b). Scan rate: 100 mV/s.
with non-imprinted PPD film modification, were prepared under the same conditions but in the absence of triclosan. 2.4. Verification of the imprinting effect The interaction between triclosan and imprinted PPD was evaluated by incubating the iPPD electrode in acetate buffer (pH 5.2) containing appropriate concentration of triclosan for 15 min under stirring. Then the films were dipped in acetate buffer for 3 min and washed by water for 15 s to remove the possible adsorptive substances on the surface. Electrochemical properties of the films were measured by cyclic voltammetry in 0.01 mol/L K4[Fe(CN)6] solution containing 1 mol/L KNO3 as the supporting electrolyte. 3. Results and discussion 3.1. Molecular imprinting electropolymerization The typical cyclic voltammograms recorded during the electropolymerization of o-PD on GC electrode in the presence of triclosan are shown in Fig. 2. The peak current drops dramatically with each scan. The oxidation wave appears completely irreversible. Finally the current approaches to zero, indicating the formation of nonconducting film, which hinders the monomer access to electrode surface. No significant difference was observed between the cyclic voltammograms obtained in the presence of triclosan and in its absence, which can be explained by the fact that triclosan does not have any electroactivity on GC electrode in the potential window
Fig. 4. Cyclic voltammograms for three different electrodes in 0.01 mol/L K4[Fe(CN)6] solutions. (a) Bare GC electrode; (b) GC electrode modified with iPPD film; (c) GC electrode modified with non-imprinted PPD film. Scan rate: 100 mV/s.
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Fig. 5. Removing time curves of template. The soaking time was: (a) 0; (b) 5; (c) 10; (d) 15; (e) 30 min. Scan rate: 100 mV/s.
chosen for the polymerization in acetate buffer (pH 5.2) [14]. As a result, the structure of triclosan is not electrochemically changed in the polymerization process. 3.2. Electrochemical characterization of MIP films In order to confirm whether triclosan has been embedded in PPD film, cyclic voltammograms of the PPD film prepared in the presence or absence of triclosan were respectively recorded in borax buffer (pH 9.2). Fig. 3 shows a typical comparison of cyclic voltammograms between the two different electrodes. In the absence of triclosan (Fig. 3b) no oxidation peak can be observed. However, in the presence of triclosan (Fig. 3a) an oxidation peak with a plateau shape can be clearly observed, which corresponds to the oxidation of triclosan and means that triclosan has been embedded in PPD film. Compared to the cyclic voltammograms of free triclosan in free in solution on bare electrode[13], oxidation peak of triclosan embedded in PPD film is more wide in shape and more positive in potential. This may be explained by the interaction between triclosan and the iPPD film, which make the electron transfer of triclosan embedded in film more difficult than triclosan free in solution. To further characterize the prepared electrodes, cyclic voltammograms of bare GC electrode, non-imprinted PPD electrode and iPPD electrode were performed in 0.01 mol/L K4[Fe(CN)6] solutions containing 1 mol/L KNO3. Fig. 4 shows a typical comparison of cyclic voltammograms among the three types of GC electrodes. The cyclic voltammetric results indicated that quasi-reversible electrochemical redox reactions of [Fe(CN)6]3−/[Fe(CN)6] 4− ion pair occurred with the bare GC electrode (Fig. 4a) with the peak potential differences of 130 mV
Fig. 6. Incubation time curves of the imprinted sensor. Incubation time was: (a) 0; (b) 5; (c) 10; (d) 15; (e) 20 min. Scan rate: 100 mV/s.
Fig. 7. A. Cyclic voltammograms of triclosan of different concentration in 0.01 mol/L K4 [Fe(CN)6] solutions containing 1 mol/L KNO3. (a) 0; (b) 8.0 × 10− 7; (c) 1.6 × 10− 6; (d) 2.4 × 10− 6; (e) 3.2 × 10− 6; (f) 4.0 × 10− 6; (g) 4.8 × 10− 6; (h) 5.6 × 10−5 mol/L. Scan rate: 100 mV/s.
and the ratio of the peak currents of about 1;1 respectively. As expected, almost no electrochemistry properties could be observed with the nonimprinted PPD electrode (Fig. 4c), which indicates that electro-inactive PPD film completely covered on the surface of the GC electrode and hindered ferrocyanide access to electrode surface. It is also noted that comparing to the bare electrode, an obvious current decrease appeared by using the electrode with iPPD film (Fig. 3b). This behavior is attributed to the presence of cavities that enable ferrocyanide diffuse through iPPD polymer towards the GC electrode, in contrast with non-imprinted PPD electrode, no redox peak can be observed. 3.3. Analytical features Triclosan can be determined by direct or indirect method using the iPPD film sensor. Direct method is based on the properties of triclosan easily producing oxidation peak through cavities existing in iPPD film in borax buffer. The determination of the oxidation peak current of triclosan is simple in operation, but the oxidation of triclosan at electrode surface is usually associated with the formation of an insulating polymeric blocking film[13]. Our experiments also found that the oxidation peak current of triclosan in iPPD electrode gradually decreases with the increase of scan times in borax buffer, which make the continuous measurement by direct method infeasible. In the indirect method, ferrocyanide is used as the mediator between the imprinted electrodes and substrate solutions containing triclosan. When the iPPD electrode was incubated in solution containing triclosan, the cavities in film were partially occupied by triclosan, which would lead to the
Fig. 8. Relative current changes in the iPPD sensor for different concentrations of triclosan and some interferents. (a) triclosan; (b) catechol; (c) 2,4,6-trichlorophenol; (d) 2,4-dichlorophenol; (e) 2,8-DCDD; (f) 1-(2-Pyridylazo)-2-naphthol.
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decrease of current signal produced by ferrocyanide. The higher the concentration of triclosan is, the lower the current would be. As is known, ferrocyanide possesses good electrochemical stability and can assure more reliable analytical results than the direct method. Indirect method was therefore preferred for the determination of triclosan. 3.3.1. Template removing treatment It is difficult to achieve a satisfactory sensitivity and reproducibility if template molecule cannot be eluted completely. Alcohol, ultra pure water and NaOH solution were respectively applied to remove template. The results indicated that alcohol and water can only partially remove the template, while NaOH solution can remove it more quickly and completely because triclosan can easily dissolve in NaOH solution. Cyclic voltammograms in 0.01 mol/L K4[Fe(CN)6] solution were performed after the iPPD electrode was dipped in NaOH solution for different time intervals. Fig. 5 shows the voltammograms corresponding to different soaking times. As can be seen, the current increased rapidly with the increase of soaking time at the beginning, but gradually approached stable after the soaking time was more than 10 min. So soaking in 0.1 mol/L NaOH solution for 10 min was chosen as the best condition for template removal. 3.3.2. Incubation time Incubation involved in the measurements is another critical factor for the performance of imprinted sensor. After template was removed from film, the iPPD sensor was incubated in acetate buffer solution (pH 5.2) containing triclosan. Fig. 6 shows the results obtained with different incubation times. The reduction peak current decreases with increasing incubation time, and a stable response is obtained after 15 min incubation. Thus, the optimum incubation time should be 15 min for the determination of triclosan. 3.3.3. The dependence of the current on the concentration of triclosan Cyclic voltammograms in 0.01 mol/L K4[Fe(CN)6] solution were performed after the iPPD electrode was incubated in solutions containing triclosan of different concentration for 15 min. Fig. 7A shows the dependence of the reduction current on the concentration of triclosan. As can be seen, the peak current decreases with the increase of triclosan concentration, due to the increasing number of the binding sites in the film were occupied by triclosan molecules. A calibration curve between the relative reduction peak current and the triclosan concentration is exhibited in Fig. 7B. Considering the absolute currents may vary with surface areas of the electrodes used and the distant between work electrode and auxiliary electrode in each experiment, relative current change (Δi/i0) was therefore suggested to indicate current response. Here, Δi=i0 −ic, i0 and ic is the current when concentration of triclosan is 0 and c mol/L respectively. As can be seen, at the high concentration range, the relative current change tend to be stable, which indicated that the imprinting sites were gradually occupied by triclosan molecules. The appearance of a platform in the curve reveals that the imprint cavities are saturated. The linear range is therefore estimated to be 2.0×10− 7 to 3.0×10− 6 mol/L and the detection limit is calculated to be 8.0×10− 8 mol/L based on the 3σ of blank signals. The linear calibration graphs of (Δi/i0) versus concentration of triclosan (c) can be described by the following equation, y=Δi/i0 =0.1079 c (µmol/L)+0.0078, and the correlation coefficient is 0.9968. 3.3.4. Selectivity of the sensor MIPs have specific selectivity towards the template molecule [27]. The specific recognition is based on the interaction between the template and the imprinting sites. To verify the specificity of iPPD sensor towards triclosan, interference experiments were performed. Compounds including catechol, 1-(2-Pyridylazo)-2-naphthol, 2,4dichlorophenol, 2,4,6-trichlorophenol and 2,8-DCDD were added into the supporting electrolyte respectively. As shown in Fig. 8, almost no interference was observed with 1-(2-Pyridylazo)-2-naphthol, which is
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larger in size than triclosan and cannot enter into the imprinting sites present in iPPD film. Only a slight interference was obtained with 2,8DCDD, which is similar to triclosan in structure, but still mismatch the imprinting sites and shows good selectivity. Comparatively, interferences produced by catechol, 2,4-dichlorophenol and 2,4,6-trichlorophenol are slightly larger than other substances, which may be explained by the fact that these compounds are more small than triclosan in molecular size and have some chances of approaching the imprinting sites. In view of above facts, the recognition sites formed in the polymerized film have the capability to distinguish target molecules through their size, shape and functional group distribution. 4. Conclusion We have combined the use of MIPs and electropolymerization to construct an electrochemical sensor that is capable of indirectly detecting triclosan. Compared to conventional direct electrochemical determination, this method can avoid the foul of easily absorbed oligomeric products formed by triclosan's oxidation on electrode surface. Optimum analytical conditions have been established, and the constructed sensor has demonstrated to have good selectivity to triclosan determination. The strategy proposed in this work is potentially applicable to the electrochemical determination of other important molecules without electrochemical activities. Acknowledgement We are grateful to acknowledge the financial support of Jiangnan University Youth Fund in China (No. 20070344). References [1] H. Singer, S. Müller, C. Tixier, L. Pillonel, Triclosan: occurrence and fate of a widely used biocide in the aquatic environment: field measurements in wastewater treatment plants, surface waters, and lake sediments, Environ. Sci. Technol. 36 (2002) 4998–5004. [2] D.W. Kolpin, M. Skopec, M.T. Meyer, E.T. Furlong, S.D. Zaugg, Urban contribution of pharmaceuticals and other organic wastewater contaminants to streams during differing flow conditions, Sci. Total Environ. 328 (2004) 119–130. [3] P.M. Thomas, G.D. Foster, Determination of nonsteroidal anti-inflammatory drugs, caffeine, and triclosan in wastewater by gas chromatography-mass spectrometry, J. Environ. Sci. Health, A 39 (2004) 1969–1978. [4] D.W. Kolpin, E.T. Furlong, M.T. Meyer, E.M. Thurman, S.D. Zaugg, L.B. Barber, H.T. Buxton, Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999–2000: A national reconnaissance, Environ. Sci. Technol. 36 (2002) 1202–1211. [5] D.E. Latch, J.L. Packer, W.A. Arnold, K. McNeil, Photochemical conversion of triclosan to 2,8-dichlorodibenzo-p-dioxin in aqueous solution, J. Photochem. Photobiol., A 158 (2003) 63–66. [6] M. Mezcua, M.J. Gómez, I. Ferrer, A. Aguera, M.D. Hernando, A.R. Fernández-Alba, Evidence of 2,7/2,8-dibenzodichloro-p-dioxin as a photodegradation product of triclosan in water and wastewater samples, Anal. Chim. Acta 524 (2004) 241–247. [7] P. Canosa, S. Morales, I. Rodriguez, E. Rubi, R. Cela, M. Gomez, Aquatic degradation of triclosan and formation of toxic chlorophenols in presence of low concentrations of free chlorine, Anal. Bioanal. Chem. 383 (2005) 1119–1126. [8] A.E. Greyshock, P.J. Vikesland, Environ, triclosan reactivity in chloraminated waters, Sci. Technol. 40 (2006) 2615–2622. [9] H.T. Rasmussen, R. McDonough, R.J. Gargiullo, B.P. McPherson, Determination of triclosan in human dental plaque by gas chromatography with atomic emission detection, J. High Resolut. Chromatogr. 19 (1996) 359–361. [10] J.L. Wu, N.P. Lama, D. Martens, A. Kettrup, Z.W. Cai, Triclosan determination in water related to wastewater treatment, Talanta 72 (2007) 1650–1654. [11] A. Sanches-Silva, R. Sendón-García, J. López-Hernández, P. Paseiro-Losada, Determination of triclosan in foodstuffs, J. Sep. Sci. 28 (2005) 65–72. [12] S.G. Chu, C.D. Metcalfe, Simultaneous determination of triclocarban and triclosan in municipal biosolids by liquid chromatography tandem mass spectrometry, J. Chromatogr., A 1164 (2007) 212–218. [13] M. Amiri, S. Shahrokhian, E. Psillakis, F. Marken, Electrostatic accumulation and determination of triclosan in ultrathin carbon nanoparticle composite film electrodes, Anal. Chim. Acta 593 (2007) 117–122. [14] R.M. Pemberton, J.P. Hart, Electrochemical behaviour of triclosan at a screenprinted carbon electrode and its voltammetric determination in toothpaste and mouthrinse products, Anal. Chim. Acta 390 (1999) 107–115. [15] P. Raghupathy, J. Mathiyarasu, James Joseph, K.L.N. Phani, V. Yegnaraman, Hydrotrope-driven disruption of micellar encapsulants for voltammetric detection of triclosan, J. Electroanal. Chem. 584 (2005) 210–214.
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