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A simple label free colorimetric method for glyphosate detection based on the inhibition of peroxidase-like activity of Cu(Ⅱ)
Sensors and Actuators B 228 (2016) 410–415
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A simple label free colorimetric method for glyphosate detection based on the inhibition of peroxidase-like activity of Cu(II) Yaqing Chang a,b , Zhe Zhang a , Jinhui Hao a,b , Wenshu Yang a,b , Jilin Tang a,∗ a b
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China University of Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e
i n f o
Article history: Received 7 September 2015 Received in revised form 7 January 2016 Accepted 13 January 2016 Available online 19 January 2016 Keywords: 3,3 ,5,5 -Tetramethylbenzidine (TMB) Colorimetric Glyphosate UV–vis Peroxidase-like activity
a b s t r a c t The detection of glyphosate (Glyp) in water is of increasing importance because of its potential danger to environmental and health. In this study, a simple and label free colorimetric method for Glyp detection is proposed based on the inhibition of peroxidase-like activity of Cu2+ . Cu2+ possesses the peroxidaselike activity that could catalyze the oxidation of 3,3 ,5,5 -tetramethylbenzidine (TMB) to oxidized TMB (oxTMB) in the presence of H2 O2 , resulting in a color change of the solution. However, the peroxidase-like activity of Cu2+ could be strongly hindered by Glyp due to the formation of Glyp–Cu2+ complexes. The color change of the solution or the absorbance change of oxTMB is utilized to monitor the concentration of Glyp. The linear range of the proposed method is 2–200 M and the detection limit is 1 M. Furthermore, as low as 10 M Glyp can be clearly distinguished by naked eyes. Also, this proposed method is successfully applied for screening Glyp in real samples and shows great promise for environmental monitoring owing to its simplicity, rapidness and high selectivity. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Glyphosate (Glyp) is a broad-spectrum, non-selective and systemic herbicide [1]. Glyp could restrain the activity of 5enolpyruvylshikimate-3-phosphate synthase, which involves in the biosynthesis of aromatic amino acids such as phenylalanine, tyrosine, and tryptophan [2]. Owing to its high herbicidal activity, in the past decades, Glyp has been widely used as a plant killer in agriculture for the control of weeds, shrubs, and grasses. However, the indiscriminate application of Glyp in agricultural production produces lots of questions regarding environmental pollution and health hazards. Many studies have shown the possible toxicological effects of Glyp to human and it may occur through contaminated drinking water and agricultural products. It has been evidenced that Glyp induces cell death through apoptotic and autophagic [3,4], affects cell cycle regulation [5], and is a potential endocrine disruptor [6]. Accordingly, monitoring the concentration of Glyp in biological and environmental samples is of great importance. The traditional methods to detect Glyp in various samples usually employ high-performance liquid chromatography (HPLC) [7–9] and gas chromatography coupled to a mass spectrometer (GC–MS)
∗ Corresponding author. Fax: +86 431 85262734. E-mail address: [email protected] (J. Tang). http://dx.doi.org/10.1016/j.snb.2016.01.048 0925-4005/© 2016 Elsevier B.V. All rights reserved.
[10–13]. However, to realize high sensitivity and specificity, derivatization procedures are usually required to make Glyp incorporate chromophores or fluorophores [14,15]. And the derivatization procedures are quite complicated and time-consuming. Other methods such as cyclic voltammetry [16], amperometry [17], capillary electrophoresis (CE) [18], enzyme-linked immunosorbent assay (ELISA) [19], and fluorescent spectrometry [20] have been developed for Glyp detection. However, cyclic voltammetry, amperometry and CE usually do not have high sensitivity and selectivity. Although ELISA could realize sensitive and accurate detection of Glyp, it suffers from the need to raise antibodies. Moreover, an expensive enzymelinked antibody is essential to a typical ELISA and the antibody is easily influenced by high temperature [21]. The fluorescent spectrometry method needs complicated synthesis or the modification of the probe. Therefore, it is imperative to develop simple, sensitive and cost-effective methods for the detection of Glyp. Alternatively, colorimetric detection has gained growing popularity on account of the obvious color change, easy operation and simple readout by the naked eye. As one of the most important transition metal, Cu2+ has been employed as a mimic peroxidase for the detection of glucose level in human blood [22], pyrophosphate activity [23] and H2 O2 [24]. Compared with horseradish peroxidase (HRP) and HRP-micking DNAzyme or nanomaterial that possess intrinsic peroxidase-like activity, Cu2+ is low cost, easy to obtain and can be
Y. Chang et al. / Sensors and Actuators B 228 (2016) 410–415
used directly. These distinguished advantages inspire us to develop a novel Cu2+ -based colorimetric method for the detection of Glyp. Herein, we present a simple, cost-effective and label-free Cu2+ -based colorimetric method for Glyp detection based on the inhibition of peroxidase-like activity of Cu2+ . Cu2+ could catalyze the oxidation of TMB in the presence of H2 O2 . However, the ability of Cu2+ to catalyze the oxidation of TMB is inhibited when Cu2+ is pre-incubated with Glyp due to the formation of Glyp–Cu2+ complexes. The color change of the oxTMB in this process could be used for the detection of Glyp. On the basis of this mechanism, Glyp in real samples have been successfully detected with high sensitivity and selectivity. The proposed method does not need any modification of probe or complicated operations, making it simple and time-saving.
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2.3. Analysis of Glyp in real samples In order to prove the feasibility of Cu2+ -based colorimetric method, the spectral responses of Glyp in real samples were recorded. The drinking water, ground water and lake water samples were obtained from the local brands Quanyangquan, our lab and the South Lake of Changchun, Jilin province, China, respectively. All the samples were filtered through a 0.22 m filtered membrane and then different concentrations of Glyp standard solutions were added into the obtained samples. The detection procedure of Glyp in real samples was in accordance with the above mentioned experiment for Glyp detection.
3. Results and discussion 3.1. The mechanism of the sensing system
2. Experimental 2.1. Reagent and chemicals Glyphosate, Copper(II) Chloride Dihydrate (CuCl2 ·2H2 O), glufosinate ammonium (GLA), dicamba, aminomethylphosphonic acid (AMPA), and 3,3 ,5,5 -tetramethylbenzidine (TMB) were purchased from Aladdin Reagent Company (Shanghai, China). Acetochlor standared solution and atrazine solution were obtained from J&K chemical Ltd. (Beijing, China). Acetic acid was a product of Sinopharm Chemical Reagent Company (Shanghai, China). H2 O2 (30 wt%), sodium salts of anion (H2 PO4 − , SO4 2− , F− , Cl− , NO3 − , C2 O4 2− , HPO4 2− and CO3 2− ), chloride salts of ion (Na+ , K+ , Ca2+ , Ba2+ and Mg2+ ), KNO3 , KBr, and Pb(NO3 )2 were obtained from Beijing Chemical Reagent Factory (Beijing, China). All other chemicals were of analytical reagent grade and directly used without further purification. Double distilled water was used throughout the experiments.
2.2. Detection of Glyp Briefly, 20 L Glyp with various concentrations and 20 L of 2.5 mM Cu2+ were incubated in 440 L of 0.1 M HAc–NaAc (pH 4.0) at room temperature for 5 min. Then 10 L of 10 mM TMB and 10 L of 1 M H2 O2 were added. Subsequently, the mixed solution was incubated in a 40 ◦ C water bath for 20 min. Afterwards, the resulting solution was measured at 652 nm by a T6 new century UV–vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China) equipped with 1.0 cm quartz cells.
Previous research has shown that Cu2+ worked as Fenton-like agent. Cu2+ can catalyze the oxidation of peroxidase substrate (TMB and 2,2 -azinobis-(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS)) in the presence of H2 O2 , leading to a colored product [22,23,25]. Cu2+ and Glyp could form soluble N-(phosphonomethyl) glycine copper(II) complexes, in which both phosphonate and carboxylate donor groups are believed to be associated with chelation at axial positions [26,27]. Along with the formation of complexes with Glyp, the redox potential of Cu2+ changes and thus hampers the Fentonlike reaction. And therefore Cu2+ has a lower efficiency of catalyzing the oxidation of peroxidase substrate [23]. Scheme 1(B) outlines the basic principle of the colorimetric detection of Glyp. In the absence of Glyp, Cu2+ exhibit mimic enzyme activity and catalyze oxidation of TMB to oxTMB and thus produce a typical blue color. The oxTMB has a maximum absorption at 652 nm, which is commonly used in the TMB-based colorimetric detection. However, when Glyp is added, Cu2+ and Glyp could form Glyp–Cu2+ complexes. The formation of complexes leads to the inhibition of the peroxidase-like activity of Cu2+ . So the adsorption of oxTMB at 652 nm decreases with the addition of Glyp. Therefore, we could detect Glyp by the variation of absorbance or directly by naked eye based on the color change of the solution. UV–vis absorption spectroscopy was investigated to prove the feasibility of the proposed colorimetric method (Fig. 1). It can be seen that the mixture of TMB and H2 O2 was colorless and produced a weak absorbance around 652 nm (curve a in Fig. 1). The addition of Glyp itself had no effects on the absorbance or the color of the TMB–H2 O2 mixture (curve d in Fig. 1). Curve b showed that after the addition of Cu2+ to the mixture of TMB–H2 O2 , the colorless solution
Scheme 1. Schematic illustrations of the Cu2+ –TMB–H2 O2 colorimetric reaction (A) and colorimetric detection of Glyp (B).
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Fig 1. UV–vis spectra of the reaction solutions. TMB–H2 O2 (a), TMB–H2 O2 –Cu2+ (b), TMB–H2 O2 –Cu2+ + Glyp (c) and TMB–H2 O2 + Glyp (d). Inset: photographic images corresponding to curve. The concentrations of Cu2+ , Glyp, TMB and H2 O2 were 100 M, 200 M, 0.2 mM and 20 mM, respectively.
Fig. 2. The plot of the A652 versus the incubation time of Cu2+ and Glyp. The concentrations of Cu2+ , Glyp, TMB and H2 O2 were 100 M, 200 M, 0.2 mM and 20 mM, respectively.
3.2. Optimization of the detection conditions turned blue and the absorbance increased compared to those in the absence of Cu2+ , indicating the mimic enzyme activity of Cu2+ . When Cu2+ was pre-incubated with Glyp and then added into the mixture of TMB-H2 O2 , the value of absorption decreased and the blue color faded (curve c in Fig. 1). The results confirmed that the peroxidase-like activity of Cu2+ was indeed hindered by Glyp owing to the formation of Glyp–Cu2+ complexes. That is to say, Glyp–Cu2+ complexes had a much lower catalytic ability. From these results, it could be concluded that the proposed method is suitable for the sensitive detection of Glyp.
To obtain higher sensitivity and wider linear range, detection conditions such as Cu2+ concentration, temperature, incubation time and pH were optimized before the application of the proposed method. Absorbance difference A652 , that is, A0 –A was used as a criterion to optimize the detection conditions. A0 and A represented the absorbance at 652 nm in the absence and presence of 100 M Glyp, respectively. In order to study the effect of the concentration of Cu2+ on A652 , we investigated ten different concentrations of Cu2+ . Fig. 3a shows that the value of A652 first increased with the concentration of
Fig. 3. Optimization of the reaction conditions for Glyp detection. Plot of A652 in the absence and presence of Glyp and A652 versus the concentration of Cu2+ (a), temperature (b), time (c) and pH (d). The concentrations of Glyp, TMB and H2 O2 were 100 M, 0.2 mM and 20 mM, respectively and the concentration of Cu2+ in (b)–(d) was 100 M.
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Fig. 4. UV–vis spectra in the presence of Glyp with various concentrations. From a–k were 0, 2, 10, 25, 50, 100, 150, 200, 300, 500 and 700 M, respectively (a). Linear calibration plot for Glyp detection. Error bars illustrated the standard deviations of three independent measurements (b). Corresponding photographs of the color change. The concentrations of Glyp from 1 to 8 were 0, 2, 10, 25, 50, 100, 150 and 200 M, respectively (c). (For interpretation of the references to color in the text, the reader is referred to the web version of this article.)
Cu2+ and then reached a maximum value when the concentration of Cu2+ was 100 M. Therefore, 100 M Cu2+ was chosen for the subsequent colorimetric detection. Temperature played an important role in most reactions and too high or too low temperature was not appropriate for the reaction. We then explored the effect of temperature on A652 . It is seen clearly from Fig. 3b, the A652 increased with the increasing temperature in the range from 20 ◦ C to 40 ◦ C. Further increased in temperature resulted in the decrease of A652 . Therefore, 40 ◦ C was selected as the optimal incubation temperature. The effect of the pre-incubation time of Cu2+ and Glyp on the Cu2+ catalysis was first examined. As shown in Fig. 2, A652 could reach a constant level in less than 5 min and therefore 5 min was chosen as the pre-incubation time. Then the influence of the reaction time for catalytic oxidation of TMB was examined. Cu2+ and Glyp were pre-incubated for 5 min before the catalytic reaction was carried out. As illustrated in Fig. 3c, A652 increased gradually with the catalytic reaction time, and came to a maximum value when reaction time was 20 min. Therefore, the incubation time of 20 min was applied to the detection system in the following experiments. The pH value was another crucial factor for most sensing system. Therefore, the effect of pH (3.0–6.0) of the buffer was investigated and the results are described in Fig. 3d. It can be obviously seen that the maximum A652 was obtained when the pH was 4.0. Accordingly, we utilized pH 4.0 in the following experiments.
Fig. 5. Selectivity of the proposed method for Glyp against other ions. 1: Glyp; 2: HPO4 2− ; 3: NO3 − ; 4: Cl− ; 5: Br− ; 6: CO3 2− ; 7: H2 PO4 − ; 8: SO4 2− ; 9: Ca2+ ; 10: Mg2+ ; 11: K+ ; 12: Pb2+ ; 13: Na+ ; 14: GLA; 15: dicamba; 16: AMPA; 17: acetochlor; 18: atrazine. The concentration of acetochlor and atrazine were 4 g/mL, and Glyp and other chemicals were 200 M. Error bars represent the standard deviations of three replicate measurements.
ranged from 2 to 200 M (R = 0.99542). The detection limit was calculated to be 1 M at a signal-to-noise ratio of 3, which was lower than the maximum contaminant level (MCL) of Glyp in drinking water set by the US Environmental Protection Agency (4.14 M) [28]. More importantly, from the photographs, the containment level of 10 M can be clearly distinguished by naked eye.
3.3. Detection of Glyp 3.4. Interference study Under the optimized conditions, we explored the catalytic ability of Cu2+ on the TMB–H2 O2 reaction after incubation with different concentrations of Glyp. As shown in Fig. 4a, with the increase of Glyp concentrations, the absorbance at 652 nm decreased and tended to constant when the concentration of Glyp was over 200 M. The corresponding color of the solution gradually changed from blue to colorless (Fig. 4c). Fig. 4b shows that the A652 exhibited a good linear relationship when the concentration of Glyp
The selectivity of this method was verified by investigating some commonly found substances in water, such as Cl− , NO3 − , Br− , SO4 2− , HPO4 2− , H2 PO4 − , Mg2+ , K+ and Ca2+ . Besides, Glyp analogs glufosinate ammonium (GLA) and aminomethylphosphonic acid (AMPA) and other compounds such as dicamba, acetochlor and atrazine were also tested. As shown in Fig. 5, only Glyp could induce a markedly increase of A652 , whereas no obvious change of
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Table 1 Application of the proposed method for detection of Glyp in real samples. Samples
Added (M)
Found (M)
Recovery (%)
RSD (n = 3,%)
Drinking water Ground water Lake water
4 50 4 50 4 50
4.17 56.31 3.69 49.64 4.64 58.69
104.2 112.6 92.3 99.3 116.1 117.4
0.55 0.51 1.66 1.4 0.85 0.55
A652 was observed in the presence of other interfering substance. The results indicated that the Cu2+ -based colorimetric method had a high selectivity toward Glyp. The excellent selectivity could be attributed to that only Glyp could form complexes with Cu2+ . 3.5. Detection of Glyp in real samples To ascertain the accuracy and applicability of the colorimetric method, Glyp in real samples was investigated. Two different concentrations of Glyp were spiked in the three water samples, respectively, and then detected with the proposed method. As listed in Table 1, the recoveries vary from 92.3% to 117.4% with the RSD of 0.51–1.66%, indicating that the proposed colorimetric method was of high accurate and could be applied for rapid detection of Glyp in real samples in a simple way. 4. Conclusions In conclusion, we successfully develop a simple, label free colorimetric method for the detection of Glyp based on the inhibition of peroxidase-like activity of Cu2+ . The peroxidase-like catalytic ability of Cu2+ is inhibited by the formation of Glyp–Cu2+ complexes. The color change of oxTMB is used to detect the concentration of Glyp by UV–vis spectrometer or just by naked eye. The linear range and detection limit were 2–200 M and 1 M, respectively. The formation of Glyp–Cu2+ complexes makes this colorimetric method show excellent selectivity to Glyp. The proposed method does not need any modification of probe or complicated operations and it is promising in detection of Glyp in real samples. For its selectivity and simplicity, we believe that the colorimetric method will have potential applications in environmental monitoring field. Acknowledgment This work was supported by the National Basic Research Program of China (973 Program; No. 2011CB935800). References [1] Y. Zhu, F. Zhang, C. Tong, W. Liu, Determination of glyphosate by ion chromatography, J. Chromatogr. A 850 (1999) 297–301. [2] H.C. Steinrücken, N. Amrhein, The herbicide glyphosate is a potent inhibitor of 5-enolpyruvylshikimic acid-3-phosphate synthase, Biochem. Biophys. Res. Commun. 94 (1980) 1207–1212. [3] É. Clair, R. Mesnage, C. Travert, G.-É. Séralini, A glyphosate-based herbicide induces necrosis and apoptosis in mature rat testicular cells in vitro, and testosterone decrease at lower levels, Toxicol. In Vitro 26 (2012) 269–279. [4] Y.-x. Gui, X.-n. Fan, H.-m. Wang, G. Wang, S.-d. Chen, Glyphosate induced cell death through apoptotic and autophagic mechanisms, Neurotoxicol. Teratol. 34 (2012) 344–349. [5] J. Marc, O. Mulner-Lorillon, R. Bellé, Glyphosate-based pesticides affect cell cycle regulation, Biol. Cell 96 (2004) 245–249. [6] C. Gasnier, C. Dumont, N. Benachour, E. Clair, M.-C. Chagnon, G.-E. Séralini, Glyphosate-based herbicides are toxic and endocrine disruptors in human cell lines, Toxicology 262 (2009) 184–191. [7] R. Vreeken, P. Speksnijder, I. Bobeldijk-Pastorova, T.H. Noij, Selective analysis of the herbicides glyphosate and aminomethylphosphonic acid in water by on-line solid-phase extraction-high-performance liquid chromatography–electrospray ionization mass spectrometry, J. Chromatogr. A 794 (1998) 187–199.
[8] K. Takahashi, M. Horie, N. Aoba, Analysis of glyphosate and its metabolite, aminomethylphosphonic acid, in agricultural products by HPLC, Shokuhin Eiseigaku Zasshi 42 (2001) 304–308. [9] F. Fang, R. Wei, X. Liu, Novel pre-column derivatisation reagent for glyphosate by high-performance liquid chromatography and ultraviolet detection, Int. J. Environ. Anal. Chem. 94 (2014) 661–667. [10] Z.-X. Guo, Q. Cai, Z. Yang, Determination of glyphosate and phosphate in water by ion chromatography-inductively coupled plasma mass spectrometry detection, J. Chromatogr. A 1100 (2005) 160–167. [11] A. Royer, S. Beguin, J. Tabet, S. Hulot, M. Reding, P. Communal, Determination of glyphosate and aminomethylphosphonic acid residues in water by gas chromatography with tandem mass spectrometry after exchange ion resin purification and derivatization. Application on vegetable matrixes, Anal. Chem. 72 (2000) 3826–3832. [12] Z.H. Kudzin, D.K. Gralak, J. Drabowicz, J. Łuczak, Novel approach for the simultaneous analysis of glyphosate and its metabolites, J. Chromatogr. A 947 (2002) 129–141. [13] E. Börjesson, L. Torstensson, New methods for determination of glyphosate and (aminomethyl) phosphonic acid in water and soil, J. Chromatogr. A 886 (2000) 207–216. [14] M.P. Abdullah, J. Daud, K.S. Hong, C.H. Yew, Improved method for the determination of glyphosate in water, J. Chromatogr. A 697 (1995) 363–369. [15] I. Durán Merás, T. Galeano Dı´ıaz, M.a. Alexandre Franco, Simultaneous fluorimetric determination of glyphosate and its metabolite, aminomethylphosphonic acid, in water, previous derivatization with NBD-Cl and by partial least squares calibration (PLS), Talanta 65 (2005) 7–14. [16] E.A. Songa, O.A. Arotiba, J.H.O. Owino, N. Jahed, P.G.L. Baker, E.I. Iwuoha, Electrochemical detection of glyphosate herbicide using horseradish peroxidase immobilized on sulfonated polymer matrix, Bioelectrochemistry 75 (2009) 117–123. [17] F. Sánchez-Bayo, R.V. Hyne, K.L. Desseille, An amperometric method for the detection of amitrole, glyphosate and its aminomethyl-phosphonic acid metabolite in environmental waters using passive samplers, Anal. Chim. Acta 675 (2010) 125–131. [18] M. Corbera, M. Hidalgo, V. Salvado, P. Wieczorek, Determination of glyphosate and aminomethylphosphonic acid in natural water using the capillary electrophoresis combined with enrichment step, Anal. Chim. Acta 540 (2005) 3–7. [19] M. Mörtl, G. Németh, J. Juracsek, B. Darvas, L. Kamp, F. Rubio, et al., Determination of glyphosate residues in Hungarian water samples by immunoassay, Microchem. J. 107 (2013) 143–151. [20] J. Guo, Y. Zhang, Y. Luo, F. Shen, C. Sun, Efficient fluorescence resonance energy transfer between oppositely charged CdTe quantum dots and gold nanoparticles for turn-on fluorescence detection of glyphosate, Talanta 125 (2014) 385–392. [21] X. Ding, K.-L. Yang, Development of an oligopeptide functionalized surface plasmon resonance biosensor for online detection of glyphosate, Anal. Chem. 85 (2013) 5727–5733. [22] A.K. Dutta, S. Das, S. Samanta, P.K. Samanta, B. Adhikary, P. Biswas, CuS nanoparticles as a mimic peroxidase for colorimetric estimation of human blood glucose level, Talanta 107 (2013) 361–367. [23] L. Zhang, M. Li, Y. Qin, Z. Chu, S. Zhao, A convenient label free colorimetric assay for pyrophosphatase activity based on a pyrophosphate-inhibited Cu2+ –ABTS–H2 O2 reaction, Analyst 139 (2014) 6298–6303. [24] J. Guan, J. Peng, X. Jin, Synthesis of copper sulfide nanorods as peroxidase mimics for colorimetric detection of hydrogen peroxide, Anal. Methods 7 (2015) 5454–5461. [25] W. He, H. Jia, X. Li, Y. Lei, J. Li, H. Zhao, et al., Understanding the formation of CuS concave superstructures with peroxidase-like activity, Nanoscale 4 (2012) 3501–3506. [26] J. Sheals, P. Persson, B. Hedman, IR and EXAFS spectroscopic studies of glyphosate protonation and copper(II) complexes of glyphosate in aqueous solution, Inorg. Chem. 40 (2001) 4302–4309. [27] P.G. Daniele, C. De Stefano, E. Prenesti, S. Sammartano, Copper(II) complexes of N-(phosphonomethyl) glycine in aqueous solution: a thermodynamic and spectrophotometric study, Talanta 45 (1997) 425–431. [28] Drinking Water Standards and Health Advisories, United States Environmental Protection Agency (USEPA), Washington, DC (2011) http:// water.epa.gov/action/advisories/drinking/upload/dwstandards2012.pdf (accessed 13.10.12).
Biographies Yaqing Chang received her BS degree from Henan Normal University in 2013, China. Currently, she is studying for master degree under the supervision of Prof. Jilin Tang in Changchun Institute of Applied Chemistry. Her research interests focus mainly on the development of colorimetric sensors. Zhe Zhang received his BS and master’s degrees from Jilin University in 2009, China. Currently, he is an assistant researcher of the State Key Laboratory of Electroanaytical Chemistry, Changchun Institute of Applied Chemistry. His research interests focus on the application of nanomaterials. Jinhui Hao received his BS degree in 2010 from Tianjin University, China. Currently, he is studying for Ph.D degree under the supervision of Prof. Jilin Tang in Changchun
Y. Chang et al. / Sensors and Actuators B 228 (2016) 410–415 Institute of Applied Chemistry. His research interests focus mainly on the application of nanomaterials in the new energy. Wenshu Yang received her BS degree from Hebei University in 2009, China. Subsequently, she received her Ph.D degree in analytical chemistry from Changchun Institute of Applied Chemistry under the supervision of Prof. Bailin Zhang in 2015. Her research interests focus on the application of nanomaterials in detection.
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Jilin Tang is a professor of the State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry. Her current research includes molecular recognition and molecular self-assembly, application of cantilever sensors and application of nanomaterials in the new energy.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A simple label free colorimetric method for glyphosate detection based on the inhibition of peroxidase-like activity of Cu(II) Yaqing Chang a,b , Zhe Zhang a , Jinhui Hao a,b , Wenshu Yang a,b , Jilin Tang a,∗ a b
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China University of Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e
i n f o
Article history: Received 7 September 2015 Received in revised form 7 January 2016 Accepted 13 January 2016 Available online 19 January 2016 Keywords: 3,3 ,5,5 -Tetramethylbenzidine (TMB) Colorimetric Glyphosate UV–vis Peroxidase-like activity
a b s t r a c t The detection of glyphosate (Glyp) in water is of increasing importance because of its potential danger to environmental and health. In this study, a simple and label free colorimetric method for Glyp detection is proposed based on the inhibition of peroxidase-like activity of Cu2+ . Cu2+ possesses the peroxidaselike activity that could catalyze the oxidation of 3,3 ,5,5 -tetramethylbenzidine (TMB) to oxidized TMB (oxTMB) in the presence of H2 O2 , resulting in a color change of the solution. However, the peroxidase-like activity of Cu2+ could be strongly hindered by Glyp due to the formation of Glyp–Cu2+ complexes. The color change of the solution or the absorbance change of oxTMB is utilized to monitor the concentration of Glyp. The linear range of the proposed method is 2–200 M and the detection limit is 1 M. Furthermore, as low as 10 M Glyp can be clearly distinguished by naked eyes. Also, this proposed method is successfully applied for screening Glyp in real samples and shows great promise for environmental monitoring owing to its simplicity, rapidness and high selectivity. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Glyphosate (Glyp) is a broad-spectrum, non-selective and systemic herbicide [1]. Glyp could restrain the activity of 5enolpyruvylshikimate-3-phosphate synthase, which involves in the biosynthesis of aromatic amino acids such as phenylalanine, tyrosine, and tryptophan [2]. Owing to its high herbicidal activity, in the past decades, Glyp has been widely used as a plant killer in agriculture for the control of weeds, shrubs, and grasses. However, the indiscriminate application of Glyp in agricultural production produces lots of questions regarding environmental pollution and health hazards. Many studies have shown the possible toxicological effects of Glyp to human and it may occur through contaminated drinking water and agricultural products. It has been evidenced that Glyp induces cell death through apoptotic and autophagic [3,4], affects cell cycle regulation [5], and is a potential endocrine disruptor [6]. Accordingly, monitoring the concentration of Glyp in biological and environmental samples is of great importance. The traditional methods to detect Glyp in various samples usually employ high-performance liquid chromatography (HPLC) [7–9] and gas chromatography coupled to a mass spectrometer (GC–MS)
∗ Corresponding author. Fax: +86 431 85262734. E-mail address: [email protected] (J. Tang). http://dx.doi.org/10.1016/j.snb.2016.01.048 0925-4005/© 2016 Elsevier B.V. All rights reserved.
[10–13]. However, to realize high sensitivity and specificity, derivatization procedures are usually required to make Glyp incorporate chromophores or fluorophores [14,15]. And the derivatization procedures are quite complicated and time-consuming. Other methods such as cyclic voltammetry [16], amperometry [17], capillary electrophoresis (CE) [18], enzyme-linked immunosorbent assay (ELISA) [19], and fluorescent spectrometry [20] have been developed for Glyp detection. However, cyclic voltammetry, amperometry and CE usually do not have high sensitivity and selectivity. Although ELISA could realize sensitive and accurate detection of Glyp, it suffers from the need to raise antibodies. Moreover, an expensive enzymelinked antibody is essential to a typical ELISA and the antibody is easily influenced by high temperature [21]. The fluorescent spectrometry method needs complicated synthesis or the modification of the probe. Therefore, it is imperative to develop simple, sensitive and cost-effective methods for the detection of Glyp. Alternatively, colorimetric detection has gained growing popularity on account of the obvious color change, easy operation and simple readout by the naked eye. As one of the most important transition metal, Cu2+ has been employed as a mimic peroxidase for the detection of glucose level in human blood [22], pyrophosphate activity [23] and H2 O2 [24]. Compared with horseradish peroxidase (HRP) and HRP-micking DNAzyme or nanomaterial that possess intrinsic peroxidase-like activity, Cu2+ is low cost, easy to obtain and can be
Y. Chang et al. / Sensors and Actuators B 228 (2016) 410–415
used directly. These distinguished advantages inspire us to develop a novel Cu2+ -based colorimetric method for the detection of Glyp. Herein, we present a simple, cost-effective and label-free Cu2+ -based colorimetric method for Glyp detection based on the inhibition of peroxidase-like activity of Cu2+ . Cu2+ could catalyze the oxidation of TMB in the presence of H2 O2 . However, the ability of Cu2+ to catalyze the oxidation of TMB is inhibited when Cu2+ is pre-incubated with Glyp due to the formation of Glyp–Cu2+ complexes. The color change of the oxTMB in this process could be used for the detection of Glyp. On the basis of this mechanism, Glyp in real samples have been successfully detected with high sensitivity and selectivity. The proposed method does not need any modification of probe or complicated operations, making it simple and time-saving.
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2.3. Analysis of Glyp in real samples In order to prove the feasibility of Cu2+ -based colorimetric method, the spectral responses of Glyp in real samples were recorded. The drinking water, ground water and lake water samples were obtained from the local brands Quanyangquan, our lab and the South Lake of Changchun, Jilin province, China, respectively. All the samples were filtered through a 0.22 m filtered membrane and then different concentrations of Glyp standard solutions were added into the obtained samples. The detection procedure of Glyp in real samples was in accordance with the above mentioned experiment for Glyp detection.
3. Results and discussion 3.1. The mechanism of the sensing system
2. Experimental 2.1. Reagent and chemicals Glyphosate, Copper(II) Chloride Dihydrate (CuCl2 ·2H2 O), glufosinate ammonium (GLA), dicamba, aminomethylphosphonic acid (AMPA), and 3,3 ,5,5 -tetramethylbenzidine (TMB) were purchased from Aladdin Reagent Company (Shanghai, China). Acetochlor standared solution and atrazine solution were obtained from J&K chemical Ltd. (Beijing, China). Acetic acid was a product of Sinopharm Chemical Reagent Company (Shanghai, China). H2 O2 (30 wt%), sodium salts of anion (H2 PO4 − , SO4 2− , F− , Cl− , NO3 − , C2 O4 2− , HPO4 2− and CO3 2− ), chloride salts of ion (Na+ , K+ , Ca2+ , Ba2+ and Mg2+ ), KNO3 , KBr, and Pb(NO3 )2 were obtained from Beijing Chemical Reagent Factory (Beijing, China). All other chemicals were of analytical reagent grade and directly used without further purification. Double distilled water was used throughout the experiments.
2.2. Detection of Glyp Briefly, 20 L Glyp with various concentrations and 20 L of 2.5 mM Cu2+ were incubated in 440 L of 0.1 M HAc–NaAc (pH 4.0) at room temperature for 5 min. Then 10 L of 10 mM TMB and 10 L of 1 M H2 O2 were added. Subsequently, the mixed solution was incubated in a 40 ◦ C water bath for 20 min. Afterwards, the resulting solution was measured at 652 nm by a T6 new century UV–vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China) equipped with 1.0 cm quartz cells.
Previous research has shown that Cu2+ worked as Fenton-like agent. Cu2+ can catalyze the oxidation of peroxidase substrate (TMB and 2,2 -azinobis-(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS)) in the presence of H2 O2 , leading to a colored product [22,23,25]. Cu2+ and Glyp could form soluble N-(phosphonomethyl) glycine copper(II) complexes, in which both phosphonate and carboxylate donor groups are believed to be associated with chelation at axial positions [26,27]. Along with the formation of complexes with Glyp, the redox potential of Cu2+ changes and thus hampers the Fentonlike reaction. And therefore Cu2+ has a lower efficiency of catalyzing the oxidation of peroxidase substrate [23]. Scheme 1(B) outlines the basic principle of the colorimetric detection of Glyp. In the absence of Glyp, Cu2+ exhibit mimic enzyme activity and catalyze oxidation of TMB to oxTMB and thus produce a typical blue color. The oxTMB has a maximum absorption at 652 nm, which is commonly used in the TMB-based colorimetric detection. However, when Glyp is added, Cu2+ and Glyp could form Glyp–Cu2+ complexes. The formation of complexes leads to the inhibition of the peroxidase-like activity of Cu2+ . So the adsorption of oxTMB at 652 nm decreases with the addition of Glyp. Therefore, we could detect Glyp by the variation of absorbance or directly by naked eye based on the color change of the solution. UV–vis absorption spectroscopy was investigated to prove the feasibility of the proposed colorimetric method (Fig. 1). It can be seen that the mixture of TMB and H2 O2 was colorless and produced a weak absorbance around 652 nm (curve a in Fig. 1). The addition of Glyp itself had no effects on the absorbance or the color of the TMB–H2 O2 mixture (curve d in Fig. 1). Curve b showed that after the addition of Cu2+ to the mixture of TMB–H2 O2 , the colorless solution
Scheme 1. Schematic illustrations of the Cu2+ –TMB–H2 O2 colorimetric reaction (A) and colorimetric detection of Glyp (B).
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Fig 1. UV–vis spectra of the reaction solutions. TMB–H2 O2 (a), TMB–H2 O2 –Cu2+ (b), TMB–H2 O2 –Cu2+ + Glyp (c) and TMB–H2 O2 + Glyp (d). Inset: photographic images corresponding to curve. The concentrations of Cu2+ , Glyp, TMB and H2 O2 were 100 M, 200 M, 0.2 mM and 20 mM, respectively.
Fig. 2. The plot of the A652 versus the incubation time of Cu2+ and Glyp. The concentrations of Cu2+ , Glyp, TMB and H2 O2 were 100 M, 200 M, 0.2 mM and 20 mM, respectively.
3.2. Optimization of the detection conditions turned blue and the absorbance increased compared to those in the absence of Cu2+ , indicating the mimic enzyme activity of Cu2+ . When Cu2+ was pre-incubated with Glyp and then added into the mixture of TMB-H2 O2 , the value of absorption decreased and the blue color faded (curve c in Fig. 1). The results confirmed that the peroxidase-like activity of Cu2+ was indeed hindered by Glyp owing to the formation of Glyp–Cu2+ complexes. That is to say, Glyp–Cu2+ complexes had a much lower catalytic ability. From these results, it could be concluded that the proposed method is suitable for the sensitive detection of Glyp.
To obtain higher sensitivity and wider linear range, detection conditions such as Cu2+ concentration, temperature, incubation time and pH were optimized before the application of the proposed method. Absorbance difference A652 , that is, A0 –A was used as a criterion to optimize the detection conditions. A0 and A represented the absorbance at 652 nm in the absence and presence of 100 M Glyp, respectively. In order to study the effect of the concentration of Cu2+ on A652 , we investigated ten different concentrations of Cu2+ . Fig. 3a shows that the value of A652 first increased with the concentration of
Fig. 3. Optimization of the reaction conditions for Glyp detection. Plot of A652 in the absence and presence of Glyp and A652 versus the concentration of Cu2+ (a), temperature (b), time (c) and pH (d). The concentrations of Glyp, TMB and H2 O2 were 100 M, 0.2 mM and 20 mM, respectively and the concentration of Cu2+ in (b)–(d) was 100 M.
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Fig. 4. UV–vis spectra in the presence of Glyp with various concentrations. From a–k were 0, 2, 10, 25, 50, 100, 150, 200, 300, 500 and 700 M, respectively (a). Linear calibration plot for Glyp detection. Error bars illustrated the standard deviations of three independent measurements (b). Corresponding photographs of the color change. The concentrations of Glyp from 1 to 8 were 0, 2, 10, 25, 50, 100, 150 and 200 M, respectively (c). (For interpretation of the references to color in the text, the reader is referred to the web version of this article.)
Cu2+ and then reached a maximum value when the concentration of Cu2+ was 100 M. Therefore, 100 M Cu2+ was chosen for the subsequent colorimetric detection. Temperature played an important role in most reactions and too high or too low temperature was not appropriate for the reaction. We then explored the effect of temperature on A652 . It is seen clearly from Fig. 3b, the A652 increased with the increasing temperature in the range from 20 ◦ C to 40 ◦ C. Further increased in temperature resulted in the decrease of A652 . Therefore, 40 ◦ C was selected as the optimal incubation temperature. The effect of the pre-incubation time of Cu2+ and Glyp on the Cu2+ catalysis was first examined. As shown in Fig. 2, A652 could reach a constant level in less than 5 min and therefore 5 min was chosen as the pre-incubation time. Then the influence of the reaction time for catalytic oxidation of TMB was examined. Cu2+ and Glyp were pre-incubated for 5 min before the catalytic reaction was carried out. As illustrated in Fig. 3c, A652 increased gradually with the catalytic reaction time, and came to a maximum value when reaction time was 20 min. Therefore, the incubation time of 20 min was applied to the detection system in the following experiments. The pH value was another crucial factor for most sensing system. Therefore, the effect of pH (3.0–6.0) of the buffer was investigated and the results are described in Fig. 3d. It can be obviously seen that the maximum A652 was obtained when the pH was 4.0. Accordingly, we utilized pH 4.0 in the following experiments.
Fig. 5. Selectivity of the proposed method for Glyp against other ions. 1: Glyp; 2: HPO4 2− ; 3: NO3 − ; 4: Cl− ; 5: Br− ; 6: CO3 2− ; 7: H2 PO4 − ; 8: SO4 2− ; 9: Ca2+ ; 10: Mg2+ ; 11: K+ ; 12: Pb2+ ; 13: Na+ ; 14: GLA; 15: dicamba; 16: AMPA; 17: acetochlor; 18: atrazine. The concentration of acetochlor and atrazine were 4 g/mL, and Glyp and other chemicals were 200 M. Error bars represent the standard deviations of three replicate measurements.
ranged from 2 to 200 M (R = 0.99542). The detection limit was calculated to be 1 M at a signal-to-noise ratio of 3, which was lower than the maximum contaminant level (MCL) of Glyp in drinking water set by the US Environmental Protection Agency (4.14 M) [28]. More importantly, from the photographs, the containment level of 10 M can be clearly distinguished by naked eye.
3.3. Detection of Glyp 3.4. Interference study Under the optimized conditions, we explored the catalytic ability of Cu2+ on the TMB–H2 O2 reaction after incubation with different concentrations of Glyp. As shown in Fig. 4a, with the increase of Glyp concentrations, the absorbance at 652 nm decreased and tended to constant when the concentration of Glyp was over 200 M. The corresponding color of the solution gradually changed from blue to colorless (Fig. 4c). Fig. 4b shows that the A652 exhibited a good linear relationship when the concentration of Glyp
The selectivity of this method was verified by investigating some commonly found substances in water, such as Cl− , NO3 − , Br− , SO4 2− , HPO4 2− , H2 PO4 − , Mg2+ , K+ and Ca2+ . Besides, Glyp analogs glufosinate ammonium (GLA) and aminomethylphosphonic acid (AMPA) and other compounds such as dicamba, acetochlor and atrazine were also tested. As shown in Fig. 5, only Glyp could induce a markedly increase of A652 , whereas no obvious change of
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Table 1 Application of the proposed method for detection of Glyp in real samples. Samples
Added (M)
Found (M)
Recovery (%)
RSD (n = 3,%)
Drinking water Ground water Lake water
4 50 4 50 4 50
4.17 56.31 3.69 49.64 4.64 58.69
104.2 112.6 92.3 99.3 116.1 117.4
0.55 0.51 1.66 1.4 0.85 0.55
A652 was observed in the presence of other interfering substance. The results indicated that the Cu2+ -based colorimetric method had a high selectivity toward Glyp. The excellent selectivity could be attributed to that only Glyp could form complexes with Cu2+ . 3.5. Detection of Glyp in real samples To ascertain the accuracy and applicability of the colorimetric method, Glyp in real samples was investigated. Two different concentrations of Glyp were spiked in the three water samples, respectively, and then detected with the proposed method. As listed in Table 1, the recoveries vary from 92.3% to 117.4% with the RSD of 0.51–1.66%, indicating that the proposed colorimetric method was of high accurate and could be applied for rapid detection of Glyp in real samples in a simple way. 4. Conclusions In conclusion, we successfully develop a simple, label free colorimetric method for the detection of Glyp based on the inhibition of peroxidase-like activity of Cu2+ . The peroxidase-like catalytic ability of Cu2+ is inhibited by the formation of Glyp–Cu2+ complexes. The color change of oxTMB is used to detect the concentration of Glyp by UV–vis spectrometer or just by naked eye. The linear range and detection limit were 2–200 M and 1 M, respectively. The formation of Glyp–Cu2+ complexes makes this colorimetric method show excellent selectivity to Glyp. The proposed method does not need any modification of probe or complicated operations and it is promising in detection of Glyp in real samples. For its selectivity and simplicity, we believe that the colorimetric method will have potential applications in environmental monitoring field. Acknowledgment This work was supported by the National Basic Research Program of China (973 Program; No. 2011CB935800). References [1] Y. Zhu, F. Zhang, C. Tong, W. Liu, Determination of glyphosate by ion chromatography, J. Chromatogr. A 850 (1999) 297–301. [2] H.C. Steinrücken, N. Amrhein, The herbicide glyphosate is a potent inhibitor of 5-enolpyruvylshikimic acid-3-phosphate synthase, Biochem. Biophys. Res. Commun. 94 (1980) 1207–1212. [3] É. Clair, R. Mesnage, C. Travert, G.-É. Séralini, A glyphosate-based herbicide induces necrosis and apoptosis in mature rat testicular cells in vitro, and testosterone decrease at lower levels, Toxicol. In Vitro 26 (2012) 269–279. [4] Y.-x. Gui, X.-n. Fan, H.-m. Wang, G. Wang, S.-d. Chen, Glyphosate induced cell death through apoptotic and autophagic mechanisms, Neurotoxicol. Teratol. 34 (2012) 344–349. [5] J. Marc, O. Mulner-Lorillon, R. Bellé, Glyphosate-based pesticides affect cell cycle regulation, Biol. Cell 96 (2004) 245–249. [6] C. Gasnier, C. Dumont, N. Benachour, E. Clair, M.-C. Chagnon, G.-E. Séralini, Glyphosate-based herbicides are toxic and endocrine disruptors in human cell lines, Toxicology 262 (2009) 184–191. [7] R. Vreeken, P. Speksnijder, I. Bobeldijk-Pastorova, T.H. Noij, Selective analysis of the herbicides glyphosate and aminomethylphosphonic acid in water by on-line solid-phase extraction-high-performance liquid chromatography–electrospray ionization mass spectrometry, J. Chromatogr. A 794 (1998) 187–199.
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Biographies Yaqing Chang received her BS degree from Henan Normal University in 2013, China. Currently, she is studying for master degree under the supervision of Prof. Jilin Tang in Changchun Institute of Applied Chemistry. Her research interests focus mainly on the development of colorimetric sensors. Zhe Zhang received his BS and master’s degrees from Jilin University in 2009, China. Currently, he is an assistant researcher of the State Key Laboratory of Electroanaytical Chemistry, Changchun Institute of Applied Chemistry. His research interests focus on the application of nanomaterials. Jinhui Hao received his BS degree in 2010 from Tianjin University, China. Currently, he is studying for Ph.D degree under the supervision of Prof. Jilin Tang in Changchun
Y. Chang et al. / Sensors and Actuators B 228 (2016) 410–415 Institute of Applied Chemistry. His research interests focus mainly on the application of nanomaterials in the new energy. Wenshu Yang received her BS degree from Hebei University in 2009, China. Subsequently, she received her Ph.D degree in analytical chemistry from Changchun Institute of Applied Chemistry under the supervision of Prof. Bailin Zhang in 2015. Her research interests focus on the application of nanomaterials in detection.
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Jilin Tang is a professor of the State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry. Her current research includes molecular recognition and molecular self-assembly, application of cantilever sensors and application of nanomaterials in the new energy.