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Study of enzyme biosensor based on carbon nanotubes modified electrode for detection of pesticides residue
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Chinese Chemical Letters 19 (2008) 592–594 www.elsevier.com/locate/cclet
Study of enzyme biosensor based on carbon nanotubes modified electrode for detection of pesticides residue Shu Ping Zhang a,b,d,*, Lian Gang Shan c, Zhen Ran Tian a, Yi Zheng a, Li Yi Shi b,d,*, Deng Song Zhang b,d a
College of Science, University of Shanghai for Science and Technology, Shanghai 200093, China b School of Material Science and Engineering, Shanghai University, Shanghai 200072, China c Jiangsu Food Science College, Jiangsu 223003, China d Nano-science Technology Center, Shanghai University, Shanghai 200444, China Received 19 November 2007
Abstract The paper describes a controllable layer-by-layer (LBL) self-assembly modification technique of multi-walled carbon nanotubes (MWNTs) and poly(diallyldimethylammonium chloride) (PDDA) towards glassy carbon electrode (GCE), Acetylcholinesterase (AChE) was immobilized directly to the modified GCE by LBL self-assembly method, the activity value of AChE was detected by using i-t technique based on the modified Ellman method. Then the composition of carbaryl were detected by the enzyme electrode with 0.01U activity value and the detection limit of carbaryl is 10 12 g L 1 so the enzyme biosensor showed good properties for pesticides residue detection. # 2008 Shu Ping Zhang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Acetylcholinesterase; Carbon nanotubes modified electrode; Layer-by-layer self-assembly; Immobilization
Biosensor technology is well suited for field monitoring organic phosphorous pesticides and carbamate pesticides [1,2]. Recently, many studies have focused on the following two techniques, one is to enhance the electrochemical properties of electrode such as the sensitivity, stability and reproducibility through the modification of work electrode [3–5], the other is to improve the efficiency of enzyme immobilization through optimization [6–8]. Multi-walled carbon nanotubes (MWNTs) have many special uses because of their attractive electronic, chemical and mechanical properties. Meanwhile, MWNTs can behave electrically as a metal or as a semiconductor due to the atomic structure. The paper describes a controllable modification technique of glassy carbon electrode (GCE) with MWNTs [9–10] and introduce a controllable direct immobilization of acetylcholinesterase (AChE) on the modified electrode. By the acctivity decreasing of immobilized AChE caused by pesticides, the composition of pesticide can be determined. Electro-deposition of MWNTs on GCE was carried out by magnetic stirring for 2 h, the potential is 1.7 V. Then the modified electrode was dipped into borate buffer solution (pH 9.18) for 15 min so that the functional groups can be
* Corresponding author at: College of Science, University of Shanghai for Science and Technology, Shanghai 200093, China. E-mail addresses: [email protected] (S.P. Zhang), [email protected] (L.Y. Shi). 1001-8417/$ – see front matter # 2008 Shu Ping Zhang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2008.03.014
S.P. Zhang et al. / Chinese Chemical Letters 19 (2008) 592–594
593
Fig. 1. SEM of MWNTs: (a) raw MWNTs; (b) after treated; (c) {PDDA/MWNTs}5 assembled to silicon slide.
negatively charged. Afterwards, by immersing the GCE in 1% PDDA solution (the ion intensity was adjusted to 0.8 mol L 1 with NaCl) and 1 mg mL 1 MWNTs solution of pH 9.18 borate buffer solution for 15 min alternately, a five-PDDA/MWNTs-bilayer can be obtained. The structures were shown in Fig. 1, raw MWNTs have different diameters and tangle together (Fig. 1a). However, by treated in concentrated nitric acid, MWNTs become purer and more homogeneous (Fig. 1b), meanwhile, homogeneous and uniform multilayer of MWNTs and PDDA was showed in Fig. 1c, it is attributed to the oxidation and good dispersion of MWNTs and the layer-by-layer assembly process with PDDA [11]. CV and amperometric i-t scanning techniques were used in TCh detection and the AChE activity value at a sweep rate of 100 mV s 1 in phosphate buffer solution with 5 10 3 mol L 1 TCh, the voltammetric characteristics of TCh on GCE and {PDDA/MWNTs}5/GCE were investigated by cyclic voltammetry at the potential range of 0–1.0 V, the anodic peak current of TCh oxidation on the {PDDA/MWNTs}5/GCE (Ipa = 54 mA) is about one time bigger than that on bare GCE(Ipa = 27 mA), and the overvoltage on the {PDDA/MWNTs}5/GCE (Epa = 0.35 V) is about a half less than that of bare GCE (Epa = 0.65 V). So this can be attributed to the electrocatalytic activity of MWNTs [16]. The amperometric i-t response of {PDDA/MWNTs}5/ED/GCE on successive step changes of TCh at applied potential 0.30 V was represented in Fig. 2. So it is clear that steady-state current rises with the addition of TCh and quickly reaches a stable value. After preparing {PDDA/MWNTs}5/ED/GCE, AChE can be immobilized directly by LBL self-assembly, it is important that a PDDA membrane was fabricated at the last surface in order to avoid the fall-off of AChE [16]. Then the activity value of AChE can be obtained according to the formulas of A = 0.133c and U = 0.075A, Hereinto, A means the value at 412 nm after 5 min of reaction between TCh and DTNB, c means the concentration of TCh. Meanwhile, it was proved that the activity of immobilized AChE in 4-PDDA/AChE bilayer was stable, reproducible and always 0.01U.
Fig. 2. Amperometric response of {PDDA/MWNTs}5 multilayer films modified GCE to various volumes of TCh.
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S.P. Zhang et al. / Chinese Chemical Letters 19 (2008) 592–594
Fig. 3. Relationship between enzyme inhibition rate and concentration range of 10 10 to 10 7 g L 1.
lg[carbaryl]. Insert: Linearity of enzyme inhibition rate and
lg[carbaryl] between the
The AChE activity can also be evaluated by current value produced by TCh oxidation since ATChCl can be hydrolyzed by AChE to produce TCh. So the oxidation current value (I1) can get smaller when the AChE activity is inhibited, it is proved that the current value (I) decreased linearly with the acctivity decreasing of immobilized AchE when 80 mL ATChCl was added to PBS, a formula for inhibition rate of AChE is as the following: Inhibition% = [(I0 I1)/I0] 100%. Hereinto, I0 was 9.93 10 6A for 0.01U activity of immobilized AChE. In different concentration solutions of pesticide, inhibition rate can be determined after the 5 min immersion of enzymatic electrode, Fig. 3 is the curves between inhibition rates and pesticide concentration, it shows a good linearity between inhibition rates and lg[carbaryl] with a detection limit of 10 12 g L 1. The recovery rate of carbaryl was obtained by determining the inhibition of AchE, In 0.01 ppm carbaryl standard solution, the recovery ranged from 93.1% to 108% with an average of 103%. So the enzymatic biosensor has a good property in the detection of pesticides. By layer-by-layer (LBL) self-assembly modification technique, {PDDA/MWNTs}5/ED/GCE can be developed and AChE can be immobilized directly to it. Meanwhile, the activity value of AChE was detected by using i-t technique based on the modified Ellman method [12]. Then carbaryl residue were detected by the enzyme biosensor with 0.01U enzyme activity, the detection limit is 10 12 g L 1 in the future, further efforts will be made to develop the enzymatic biosensor and the biosensor will has a good prospect in pesticides detecting and monitoring. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
M. Trojanowicz, Electroanal. J. 14 (2002) 1311. J.C. Vidal, S. Esteban, J. Gil, et al. Talanta J. 68 (2006) 791. K. Wu, J. Fei, S. Hu, Anal. Biochem. J. 318 (2003) 100. A. Artyukhin, O. Bakajin, P. AStroeve, Langmuir J. 20 (2004) 1442. H. Kong, P. Luo, C. Gao, Polym. J. 46 (2005) 2472. R. Saini, I. Chiang, H. Peng, Am. Chem. Soc. J. 125 (2003) 3617. S. Barreira, V. Garcyia, C.J. Pereira, Phys. Chem. B J. 108 (2004) 17973. E. Katz, I. Willner, Chem. Phys. Chem. J. 5 (2004) 1084. G. Liu, Y. Lin, Electrochem. Commun. J. 8 (2006) 251. H. Zhao, J. Huang, Anal. Biochem. J. 350 (2006) 138. Y.H. Zhu, Z.L. Zhang, W. Zhao, D.W. Pang, Sensors and Actuators B J. 119 (2006) 308. G. Ellman, Arch. Biochem. Biophys. J. 74 (1958) 443.
Chinese Chemical Letters 19 (2008) 592–594 www.elsevier.com/locate/cclet
Study of enzyme biosensor based on carbon nanotubes modified electrode for detection of pesticides residue Shu Ping Zhang a,b,d,*, Lian Gang Shan c, Zhen Ran Tian a, Yi Zheng a, Li Yi Shi b,d,*, Deng Song Zhang b,d a
College of Science, University of Shanghai for Science and Technology, Shanghai 200093, China b School of Material Science and Engineering, Shanghai University, Shanghai 200072, China c Jiangsu Food Science College, Jiangsu 223003, China d Nano-science Technology Center, Shanghai University, Shanghai 200444, China Received 19 November 2007
Abstract The paper describes a controllable layer-by-layer (LBL) self-assembly modification technique of multi-walled carbon nanotubes (MWNTs) and poly(diallyldimethylammonium chloride) (PDDA) towards glassy carbon electrode (GCE), Acetylcholinesterase (AChE) was immobilized directly to the modified GCE by LBL self-assembly method, the activity value of AChE was detected by using i-t technique based on the modified Ellman method. Then the composition of carbaryl were detected by the enzyme electrode with 0.01U activity value and the detection limit of carbaryl is 10 12 g L 1 so the enzyme biosensor showed good properties for pesticides residue detection. # 2008 Shu Ping Zhang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Acetylcholinesterase; Carbon nanotubes modified electrode; Layer-by-layer self-assembly; Immobilization
Biosensor technology is well suited for field monitoring organic phosphorous pesticides and carbamate pesticides [1,2]. Recently, many studies have focused on the following two techniques, one is to enhance the electrochemical properties of electrode such as the sensitivity, stability and reproducibility through the modification of work electrode [3–5], the other is to improve the efficiency of enzyme immobilization through optimization [6–8]. Multi-walled carbon nanotubes (MWNTs) have many special uses because of their attractive electronic, chemical and mechanical properties. Meanwhile, MWNTs can behave electrically as a metal or as a semiconductor due to the atomic structure. The paper describes a controllable modification technique of glassy carbon electrode (GCE) with MWNTs [9–10] and introduce a controllable direct immobilization of acetylcholinesterase (AChE) on the modified electrode. By the acctivity decreasing of immobilized AChE caused by pesticides, the composition of pesticide can be determined. Electro-deposition of MWNTs on GCE was carried out by magnetic stirring for 2 h, the potential is 1.7 V. Then the modified electrode was dipped into borate buffer solution (pH 9.18) for 15 min so that the functional groups can be
* Corresponding author at: College of Science, University of Shanghai for Science and Technology, Shanghai 200093, China. E-mail addresses: [email protected] (S.P. Zhang), [email protected] (L.Y. Shi). 1001-8417/$ – see front matter # 2008 Shu Ping Zhang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2008.03.014
S.P. Zhang et al. / Chinese Chemical Letters 19 (2008) 592–594
593
Fig. 1. SEM of MWNTs: (a) raw MWNTs; (b) after treated; (c) {PDDA/MWNTs}5 assembled to silicon slide.
negatively charged. Afterwards, by immersing the GCE in 1% PDDA solution (the ion intensity was adjusted to 0.8 mol L 1 with NaCl) and 1 mg mL 1 MWNTs solution of pH 9.18 borate buffer solution for 15 min alternately, a five-PDDA/MWNTs-bilayer can be obtained. The structures were shown in Fig. 1, raw MWNTs have different diameters and tangle together (Fig. 1a). However, by treated in concentrated nitric acid, MWNTs become purer and more homogeneous (Fig. 1b), meanwhile, homogeneous and uniform multilayer of MWNTs and PDDA was showed in Fig. 1c, it is attributed to the oxidation and good dispersion of MWNTs and the layer-by-layer assembly process with PDDA [11]. CV and amperometric i-t scanning techniques were used in TCh detection and the AChE activity value at a sweep rate of 100 mV s 1 in phosphate buffer solution with 5 10 3 mol L 1 TCh, the voltammetric characteristics of TCh on GCE and {PDDA/MWNTs}5/GCE were investigated by cyclic voltammetry at the potential range of 0–1.0 V, the anodic peak current of TCh oxidation on the {PDDA/MWNTs}5/GCE (Ipa = 54 mA) is about one time bigger than that on bare GCE(Ipa = 27 mA), and the overvoltage on the {PDDA/MWNTs}5/GCE (Epa = 0.35 V) is about a half less than that of bare GCE (Epa = 0.65 V). So this can be attributed to the electrocatalytic activity of MWNTs [16]. The amperometric i-t response of {PDDA/MWNTs}5/ED/GCE on successive step changes of TCh at applied potential 0.30 V was represented in Fig. 2. So it is clear that steady-state current rises with the addition of TCh and quickly reaches a stable value. After preparing {PDDA/MWNTs}5/ED/GCE, AChE can be immobilized directly by LBL self-assembly, it is important that a PDDA membrane was fabricated at the last surface in order to avoid the fall-off of AChE [16]. Then the activity value of AChE can be obtained according to the formulas of A = 0.133c and U = 0.075A, Hereinto, A means the value at 412 nm after 5 min of reaction between TCh and DTNB, c means the concentration of TCh. Meanwhile, it was proved that the activity of immobilized AChE in 4-PDDA/AChE bilayer was stable, reproducible and always 0.01U.
Fig. 2. Amperometric response of {PDDA/MWNTs}5 multilayer films modified GCE to various volumes of TCh.
594
S.P. Zhang et al. / Chinese Chemical Letters 19 (2008) 592–594
Fig. 3. Relationship between enzyme inhibition rate and concentration range of 10 10 to 10 7 g L 1.
lg[carbaryl]. Insert: Linearity of enzyme inhibition rate and
lg[carbaryl] between the
The AChE activity can also be evaluated by current value produced by TCh oxidation since ATChCl can be hydrolyzed by AChE to produce TCh. So the oxidation current value (I1) can get smaller when the AChE activity is inhibited, it is proved that the current value (I) decreased linearly with the acctivity decreasing of immobilized AchE when 80 mL ATChCl was added to PBS, a formula for inhibition rate of AChE is as the following: Inhibition% = [(I0 I1)/I0] 100%. Hereinto, I0 was 9.93 10 6A for 0.01U activity of immobilized AChE. In different concentration solutions of pesticide, inhibition rate can be determined after the 5 min immersion of enzymatic electrode, Fig. 3 is the curves between inhibition rates and pesticide concentration, it shows a good linearity between inhibition rates and lg[carbaryl] with a detection limit of 10 12 g L 1. The recovery rate of carbaryl was obtained by determining the inhibition of AchE, In 0.01 ppm carbaryl standard solution, the recovery ranged from 93.1% to 108% with an average of 103%. So the enzymatic biosensor has a good property in the detection of pesticides. By layer-by-layer (LBL) self-assembly modification technique, {PDDA/MWNTs}5/ED/GCE can be developed and AChE can be immobilized directly to it. Meanwhile, the activity value of AChE was detected by using i-t technique based on the modified Ellman method [12]. Then carbaryl residue were detected by the enzyme biosensor with 0.01U enzyme activity, the detection limit is 10 12 g L 1 in the future, further efforts will be made to develop the enzymatic biosensor and the biosensor will has a good prospect in pesticides detecting and monitoring. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
M. Trojanowicz, Electroanal. J. 14 (2002) 1311. J.C. Vidal, S. Esteban, J. Gil, et al. Talanta J. 68 (2006) 791. K. Wu, J. Fei, S. Hu, Anal. Biochem. J. 318 (2003) 100. A. Artyukhin, O. Bakajin, P. AStroeve, Langmuir J. 20 (2004) 1442. H. Kong, P. Luo, C. Gao, Polym. J. 46 (2005) 2472. R. Saini, I. Chiang, H. Peng, Am. Chem. Soc. J. 125 (2003) 3617. S. Barreira, V. Garcyia, C.J. Pereira, Phys. Chem. B J. 108 (2004) 17973. E. Katz, I. Willner, Chem. Phys. Chem. J. 5 (2004) 1084. G. Liu, Y. Lin, Electrochem. Commun. J. 8 (2006) 251. H. Zhao, J. Huang, Anal. Biochem. J. 350 (2006) 138. Y.H. Zhu, Z.L. Zhang, W. Zhao, D.W. Pang, Sensors and Actuators B J. 119 (2006) 308. G. Ellman, Arch. Biochem. Biophys. J. 74 (1958) 443.