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��ࡱ�>��	�����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������'`	�R�}bjbj���?*������"�������       4X"X"X"8�"�|$L4l5��$�$�$�$�$�%|Q+U-O4Q4Q4Q4Q4Q4Q4$�6hf9Xu4� Y.�%�%Y.Y.u4  �$�$�&5c1c1c1Y.� �$ �$O4c1Y.O4c1c1  c1�$�$@7p�+��X"/Hc144<50l5c1�:_0��:c1c1��: �14Y.Y.c1Y.Y.Y.Y.Y.u4u4�0dY.Y.Y.l5Y.Y.Y.Y.444$X"444X"444      ����Acetylcholinesterase biosensor based on poly(diallyldimethylammonium chloride)-multi-walled carbon nanotubes-graphene hybrid film
Xia Sun*, Zhili Gong, Yaoyao Cao, Xiangyou Wang*
School of Agriculture and Food Engineering, Shandong University of Technology, NO.12, Zhangzhou Road, Zibo 255049, Shandong Province, P.R. China
*Corresponding author: associate professor Xia Sun and Professor XiangYouWang, Tel.:+86-533-2786558; E-mail:sunxia2151@sina.com;  HYPERLINK "mailto:wlj@cau.edu.cn" wxy@sdut.edu.cn
Abstract: A novel amperometric acetylcholinesterase (AChE) biosensor for quantitative determination of carbaryl was reported. This biosensor was fabricated by firstly immobilizing poly (diallyldimethylammonium chloride) (PDDA)-multi-walled carbon nanotubes (MWCNTs)-graphene (GR) hybrid film onto the glassy carbon electrode (GCE) surface, then AChE was immobilized onto the modified GCE to fabricate an AChE biosensor. The morphologies and electrochemistry properties of the PDDA-MWCNTs-GR hybrid were investigated by using scanning electron microscopy (SEM) and electrochemical techniques including cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), respectively. All variables involved in the preparation and analytical performance of the biosensor was optimized. The inhibition of carbaryl on the activity of AChE was proportional to carbaryl concentration in the ranges from 0.5 to 50 ng/mL and from 50 to 3000 ng/mL with the correlation coefficients of 0.9971 and 0.9968, respectively. The detection limit was calculated to be about 0.13 ng/mL. The biosensor exhibited a wide range, low detection limit, good reproducibility and high stability. Moreover, the biosensor could be used for direct analysis of practical samples, which provided a new promising tool for pesicide analysis.
Keywords: Biosensor; Acetylcholinesterase; Multi-walled carbon nanotubes; graphene; Poly (diallyldimethylammonium chloride)
Introduction
Carbaryl, one of the carbamate pesticides, has been widely used in agriculture because of its high effectiveness for insect control (Miwa et al. 2006; Arduini et al. 2006; Perez-Lopez et al. 1994). However, long-term accumulations in environment, carbaryl will present a serious risk to human health due to its high toxicity to acetylcholinesterase (AChE), a key enzyme for the function of the central nervous system in humans (Guan et al. 2012; Abad et al. 1998; Du et al. 2008; Vakurov et al. 2005; Du et al. 2008; Sattarahmady et al. 2010). Traditional analytical methods, such as liquid chromatography with ultraviolet diode array (Pietrogrande et al. 1985; McGarvey 1993), fluorescence spectrometry (De Kok et al. 1992) and mass spectrometry often coupled with mass-selective detectors, are obviously time-consuming and expensive. Thus, the development of efficient, sensitive and simple analytical method for the determination of pesticide residues in food has become more and more important.
Enzymatic method has some advantages such as rapid response, time saving, low cost and high sensitivity (Sun et al. 2010; Du et al. 2010). Among these, amperometric AChE biosensors based on the inhibition action of pesticides on AChE have shown satisfactory results, in which the enzymatic activity is employed as an indicator of quantitative measurement of insecticides (Shi et al. 2006; Kok et al. 2004; Amine et al. 2006). The performance of the biosensors depends on the effective immobilization of enzyme onto the surface of electrode. To maintain the inherent nature of enzyme, it is necessary to choose adequate supporting materials and immobilization methods (Du et al. 2007; Sotiropoulou et al. 2005; Shan et al. 2007; Zhao et al. 2009).
Recently, the use of multi-walled carbon nanotubes (MWCNTs) and graphene (GR) for the fabrication of electrochemical biosensors has attracted considerable attention (Iijima 1991; Rong et al. 2007; Wang et al. 2009; Shan et al. 2009; Kang et al. 2009). MWCNTs and GR have advantages in the biosensor field because of their unique physicochemical properties including high surface area, excellent electric conductivity, strong mechanical strength, good adsorptive ability and excellent biocompatibility (Sun et al. 2012; Li et al. 2011; Huang, Xing et al. 2011; Geim et al. 2007; Huang, Lin et al. 2011). Moreover, studies revealed that nanohybrid often can combine the merits of each component and exhibit enhanced properties (Zhang et al. 2007; Yang et al. 2009; Xiao et al. 2008). Since MWCNTs and GR are very attractive in electroanalysis, combining between them is exciting. Studies proved that MWCNTs-GR hybrid could generate synergy effect and enhance their performance in applications of biosensor (Zhang et al. 2006; Zhou et al. 2008). MWCNTs-GR hybrid can greatly facilitate heterogeneous electron-transfer processes and provide a promising electrochemical sensing platform (Zhou et al. 2008). It is also demonstrated that the electrochemical biosensor based on MWCNTs-GR hybrid modified electrode exhibited excellent analytical performance for determination of acetaminophen with a low detection limit and a wide linear range (Chen et al. 2012). However, both MWCNTs and GR are hydrophobic and tend to form agglomerates, limiting their further biological applications (Wang et al. 2008; Manso et al. 2008). So we need to find effectual dispersants to solve this problem.
Poly (diallyldimethylammonium chloride) (PDDA), a water soluble cationic polyelectrolyte, can improve the dispersibility of MWCNTs in water and form the thin films of MCNTs for various applications (Kim, Park et al. 2003; Kim, Sigmund et al. 2003; Rouse et al. 2003; Ma et al. 2004; Artyukhin et al. 2004). Additionally, PDDA has excellent binding capability with graphene and could maintain the electronic structure of graphene (Liu et al. 2010). Meanwhile, the solubility of PDDA functionalized graphene (PDDA-G) is greatly increased and can be used to construct biosensor (Feng et al. 2012). Therefore, combining MWCNTs-GR hybrid with PDDA and using PDDA-MWCNTs-GR hybrid in the AChE biosensor are worthwhile. However, to the best of our knowledge, there is no report based on PDDA-MWCNTs-GR hybrid modified electrodes for the quantitative determination of carbaryl.
In this study, we firstly presented the preparation and characterization of PDDA-MWCNTs-GR hybrid by a simple synthetic method, in which positively charged PDDA binded with negatively charged MWCNTs and GR to prevent the aggregation through electrostatic repulsion. Subsequently, the PDDA-MWCNTs-GR hybrid modified electrode for immobilization of AChE, leading to a stable AChE sensor for rapid determination of carbaryl. All variables involved in the experiment and the performance of the biosensor were investigated in detail.
Experimental
Reagents
Acetylcholinesterase (Type C3389, 500 U/mg from electric eel), acetylthiocholine chloride (ATCl), Poly (diallydimethylammonium chloride) (PDDA) (35%, w/w in water) and pralidoxime iodide were purchased from Sigma (St. Louis, USA). Carboxylated MWCNTs was obtained from Nanotech Port Co. (Shenzhen, China). GR was from Nanoon (Hebei, China). 0.1 M pH 7.0 phosphate buffer solutions (PBS) were prepared by mixing the stock solutions of NaH2PO4 and Na2HPO4. Carbaryl was standard product. Ethanol and other reagents were of analytical grade. The carbaryl solution was prepared by a step-dilution method.
Apparatus
A three-electrode system was employed with the modified GCE (d = 3 mm) as the working electrode, a platinum electrode as the auxiliary electrode and saturated calomel electrode (SCE) as the reference electrode. Electrochemical measurements were performed with CHI660D electro-chemical workstation (Shanghai Chenhua Co., China).
Preparation of biosensor
Preparation of PDDA-MWCNTs-GR hybrid
PDDA-MWCNTs-GR hybrid was prepared according to the literatures with minor modifications (Chen et al. 2012; Egu�laz et al. 2011). 2 mg of MWCNTs and GR (3:1 MWCNTs:GR) were dispersed in 4 mL of a 0.25% PDDA aqueous solution containing 0.5 M NaCl and sonicated for 30 min. The resulting dispersion was centrifuged at 14000 rpm and washed three times with water. Finally, the obtained hybrid was dispersed in some water to form a concentration of 1 mg/mL and the resulting solution was sonicated for 5 min before use.
Configuration of the biosensor
First, the bare GCE was polished to a mirror-like surface with 0.3 and 0.05 �m alumina powder, respectively, then sonicated in doubly distilled water, ethanol and doubly distilled water for 3 min in sequence and dried with nitrogen. Then 7 �L of PDDA-MWCNTs-GR suspension was dropped onto the GCE surface and dried at room temperature. Finally, the modified electrode was coated with 5.0 �L AChE solution (100 mU) to obtain the AChE/PDDA-MWCNTs-GR/GCE. The obtained biosensor was stored at 4�C when not in use. The stepwise fabrication process of the biosensor was shown in Fig. 1.
Insert Fig. 1.
Measurement procedure
For the measurement of carbaryl, the prepared AChE/PDDA-MWCNTs-GR /GCE was first immersed in pH 7.0 PBS (0.1 M) containing different concentrations of standard carbaryl solution for 12 min. Then, the AChE/PDDA-MWCNTs-GR/GCE was transferred into the electrochemical cell of 3 mL pH 7.0 PBS (0.1 M) containing 1.0 mM ATCl to study the electrochemical response by differential pulse voltammetry (DPV) technique. The inhibition was calculated as follows:
Inhibition (%) = (IP,control " IP,exp) / IP,control�100%
Where, iP, control was the peak current at the AChE/PDDA-MWCNTs-GR/GCE without carbaryl inhibition and iP, exp was the peak current at the AChE/PDDA-MWCNTs-GR/GCE with carbaryl inhibition.
After inhibited by pesticides, the AChE/PDDA-MWCNTs-GR/GCE was reactivated by washing with 0.1 M pH 7.0 PBS and immersed into 5.0 mM pralidoxime iodide for 12 min, then transferred to 0.1 M pH 7.0 PBS containing 1 mM ATCl to study the electrochemical response by DPV technique. The reactivation efficiency was calculated as follows:
R (%) = (Ir " IP,exp) / (IP,control " IP,exp) �100%
Where, Ir was the peak current at the AChE/PDDA-MWCNTs-GR/GCE after 5.0 mM pralidoxime iodide reactivation.
Preparation and determination of real samples
For the analysis of carbaryl in real samples, cleaned cabbage, garland chrysanthemum, leek and pakchoi were cut into small pieces. 5 g of each sample was sprayed with different concentrations of carbaryl and stored at 4 �C for 24 h, then mixed with 10 mL mixed solution of acetone/0.1 M pH 7.0 PBS (1/9, v/v). After that, the mixture were sonicated for 15 min and centrifuged (10 min, 10,000 rpm). The DPV analysis was performed directly in the acquired supernatants without extraction or preconcentration. The content of carbaryl in the samples can be achieved from the calibration curve.
Results and discussion
Characterization of MWCNTs, GR and PDDA-MWCNTs-GR hybrid
The morphologies of only MWCNTs and GR film together with PDDA-MWCNTs-GR hybrid film were characterized with scanning electron microscope (SEM). As shown in Fig. 2A, PDDA-MWCNTs presented a large number of filamentous structures. Fig. 2B showed a general view of PDDA-graphene which clearly illustrated the flake-like shapes of graphene. Fig. 2C showed a typical SEM image of PDDA-MWCNTs-GR hybrid film in which MWCNTs were uniformly dispersed throughout GR film. We can found that the number of junctions and extent of stacking between MWCNTs and GR in the hybrid film were both reduced compared to the only MWCNTs film (Fig. 2A) and GR film (Fig. 2B). The usual aggregation and stacking between individual MWCNTs or GR were clearly reduced due to the combining between MWCNTs and GR. This interesting property result in enhanced surface area and porosity, which are very beneficial to the further application in bioseneor field (Wang, Li et al. 2008; Walcarius et al. 2008)
Insert Fig. 2.
Electrochemical impedance analysis
Electrochemical impedance spectroscopy (EIS) is a high effective method for probing the interface properties of the modified electrodes. The EIS includes a semicircular part at higher frequencies corresponding to the electron transfer limited process and a linear part at lower frequencies corresponding to the diffusion process. The semicircle diameter of the EIS equals the interfacial electron transfer resistance (Rct). Fig. 3 showed some nyquist diagrams obtained from bare GCE (curve a), PDDA-GR/GCE (curve b), PDDA-MWCNTs/GCE (curve c), PDDA-MWCNTs -GR/GCE (curve d) and AChE/PDDA-MWCNTs-GR/GCE (curve e) using 5.0 mM [Fe(CN)6]3-/4- with 0.1 M KCl as the redox probe. The Rct of the bare GCE was 486 � (curve a), by contrast, the Rct of PDDA-GR/GCE (curve b) and PDDA-MWCNTs /GCE (curve c) decreased dramatically to about 180, 120 � respectively. This phenomenon suggested that PDDA-GR and PDDA-MWCNTs hybrid greatly improved the conductivity of the modified electrode due to their high surface area and excellent electric conductivity. For PDDA-MWCNTs-GR/GCE, the Rct was 50� (curve d), further decreased compared with curve b and c. It was attributed to the nanohybrid of MWCNTs and GR reduced the aggregation and stacking between MWCNTs or GR, which led enhanced surface area and wide porous structure of MWCNTs-GR hybrid (Chen et al. 2012). When AChE was immobilized onto the surface of PDDA-MWCNTs-GR/GCE, the Rct remarkably increased to 1100 � (curve e) due to the increase of the thickness of the interface, which insulated the conductive support and reduced the electron transfer between anionic [Fe(CN)6] 3-/4- and GCE (Zhang et al. 2009; Du et al. 2008; Chauhan et al. 2011)
Insert Fig. 3.
Cyclic voltammetric behavior of ATCl
Fig. 4A showed the cyclic voltammograms (CVs) of different fabricated electrodes in absence and presence of ATCl in 0.1 M PBS (pH 7.0). No peak was observed at bare GCE (curve a) and AChE/PDDA-MWCNTs-GR/GCE (curve b) in pH 7.0 PBS without ATCI. CV of AChE/PDDA-MWCNTs-GR/GCE (curve d) showed an irreversible oxidation peak in the presence of 1.0 mM ATCl, whereas no detectable signal was observed for PDDA-MWCNTs-GR/GCE (curve c). Conspicuously this peak ascribed to the oxidation of thiocholine, hydrolysis product of ATCl, catalyzed by immobilized AChE. The peak current produced by the oxidation of thiocholine was used as an indicator for quantitative measurement of the enzyme activity, which could reflect the biological effect of carbaryl involved in the inhibition action.
To furthet study the different modified materials of electrode, the CVs of AChE/GCE (curve a), AChE/PDDA-GR/GCE (curve b), AChE/PDDA-MWCNTs/ GCE (curve c) and AChE/PDDA-MWCNTs-GR/GCE (curve d) in the presence of 1.0 M ATCl in 0.1 M PBS (pH 7.0) were showed in Fig. 4B. AChE/GCE (curve a) showed a small peak current, which may be due to the low enzyme loading arising from the weak combination between AChE and GCE. Compared with AChE/GCE, the peak currents increased sharply on the electrode with PDDA-GR or PDDA-MWCNTs. It was estimated that PDDA-GR and PDDA-MWCNTs had the ability to promote the electron transfer. Moreover, the conductive property of PDDA-MWCNTs was better than PDDA-GR. The other reason would be the composites possessed a relatively large specific surface area, which greatly enhanced the immobilized amount of AChE. A significant increase of peak current was obtained at AChE/PDDA-MWCNTs-GR/GCE (curve d), which was attributed to the synergistic effect between PDDA-GR and PDDA-MWCNTs.
Insert Fig. 4.
Optimization of experimental parameters
Influence of PDDA-MWCNTs-GR concentration
The effect of PDDA-MWCNTs-GR concentration on the response current was studied in 0.1 M PBS (pH 7.0) containing 1.0 mM ATCl. As shown in Fig. 5A, the signal current increased gradually with the increase of PDDA-MWCNTs-GR concentration, and when it increased to 1 mg/mL, no obvious enhancement of current was observed. Since PDDA-MWCNTs-GR possessed high surface area and excellent electric conductivity, PDDA-MWCNTs-GR-coated GCE usually has better properti than bare GCE. In this study, 1 mg/mL PDDA-MWCNTs-GR was selected to prepare the biosensor.
Influence of enzyme amount
Fig. 4B showed the influence of enzyme amount on the response of biosensor. With the increasing of AChE loading amount, the steady-state current increased sharply and reached the maximal value at 0.1 U. After that, the amperometric response decreased gradually as the amount of AChE further increased. Therefore, 0.1 U of AChE was chosen for preparation of the biosensor.
Influence of detection solution pH
The sensitivity and stability of the biosensor depended greatly on the pH of electrolyte solution. Fig. 5C showed the plot of peak current of the biosensor versus different pH in 0.1 M PBS in the presence of 1.0 mM ATCl. The maximum peak current appeared at pH 7.0, which was closed to that previous report for free AChE (Bernabei et al. 1993), suggesting the immobilization did not change the fundamental microenvironment of AChE in a solution. Thus, pH 7.0 was selected for the subsequent experiment.
Influence of incubation time on inhibition
The incubation time was an important factor for capturing carbaryl. As shown in Fig. 5D, the steady-state current increased sharply with an increase of incubation time up to 12 min and then it trended to a stable value. However, the maximum value of inhibition of carbaryl was not 100%. It can be explained that the binding sites between pesticides and enzymes could reach an equilibration state (Du et al. 2010). Thus, the time of 12 min was selected as the optimum inhibition time.
Insert Fig. 5.
Detection of carbaryl
Fig. 6A showed the DPVs of the biosensor before and after incubation in various concentrations of carbaryl under the optimal conditions, the produced current decreased drastically with increasing concentration of carbaryl. It may be due to the fact that carbaryl exhibited fairly high toxicity and involved in the irreversible inhibition action on AChE, thus reduced the enzymatic activity to its substrate. The peak current changes observed in Fig. 6A reflected the activity of immobilized enzyme, which provided a simple method for determination of carbaryl. Under the optimal experimental conditions, the inhibitions of carbaryl on AChE/PDDA- MWCNTs-GR/GCE were proportional to its concentrations in two ranges, from 0.5 to 50 ng/mL and from 50 to 3000 ng/mL (Fig.6 B), the regression equation was I% = 0.313c+20.228 and I% = 0.0128c+36.185, with the correlation coefficients of 0.9971 and 0.9968, respectively. The detection limit was calculated to be 0.13 ng/mL (S/N = 3).
A comparison of analytical characteristics of AChE/PDDA-MWCNTs-GR/GCE with other reported biosensors for the detection of carbaryl was summarized in Table 1.
Insert Fig. 6.
Insert Table 1.
Precision, stability and reactivation
The inter-assay precision was estimated at 5 different electrodes for the determinations to 1.0 mM ATCl after being treated with 10 ng/mL carbaryl solutions for 12 min. Similarly, the intra-assay precision of the biosensor was evaluated by analyzing carbaryl levels for 5 replicate measurements at one enzyme electrode. The RSD values of inter-assay and intra-assay were found to be 4.1% and 4.7%, respectively, indicating acceptable precision and replicability. The enzyme electrode was stored at 4 �C in dry condition for 7 days, and no obvious decrease in the response of ATCl was observed. After a 30-day storage period, the biosensor still retained 87% of its initial current response, indicating satisfactory stability.
According to previous reports, the inhibited AChE could be reactivated by oximes, such as pralidoxime iodide (Du et al. 2007; Yin et al. 2009). After reactivation with 5.0 mM pralidoxime iodide solution for 12 min, the inhibited AChE could be regenerated more than 93% of its original activity, indicating acceptable reproducibility.
Real samples analysis
In order to evaluate the practicality of the proposed biosensor, it was evaluated by processing real samples. We performed the recovery tests by adding different concentrations of carbaryl into cabbage, garland chrysanthemum, leek and pakchoi, summarized as Table 2. The recoveries were from 92.3% to 107%, suggesting that the proposed biosensor could be used for direct analysis of relevant samples.
Insert Table 2.
Conclusions
In this work, we reported a sensitive and efficient biosensor, AChE/PDDA-MWCNTs-GR/GCE, for the detection of carbaryl. The MWCNTs-GR nanohybrid could significantly reduce the aggregation and stacking between MWCNTs or GR, which resulted in enhanced surface area and wide porous structure of the hybrid. At the same time, PDDA increased the solubility of MWCNTs-GR hybrid. Due to these good properties, the AChE/PDDA-MWCNTs-GR/GCE exhibited excellent performances for the detection of carbaryl, such as wide linear range, low detection limit, high sensitivity, good stability and acceptable reproducibility. In addition, the biosensor could be applied for direct analysis of practical samples, which would be a new promising tool for pesticides analysis. More importantly, PDDA-MWCNTs-GR hybrid provided an excellent material for fabrication of other sensors.
Acknowledgments 
This work was supported by the National Natural Science Foundation of China (No.30972055, 31101286), Agricultural Science and Technology Achievements Transformation Fund Projects of the Ministry of Science and Technology of China (No.2011GB2C60020) and Shandong Provincial Natural Science Foundation, China (No.Q2008D03).
References
Abad, J.M. Pariente, F. Hern�ndez, L. Abruna, H.D. and Lorenzo, E. 1998. Determination of organophosphorus and carbamate pesticides using a piezoelectric biosensor. Anal. Chem. 70: 2848-2855.
Amine, A. Mohammadi, H. Bourais, I. and Palleschi, G. 2006. Enzyme inhibition-based biosensors for food safety and environmental monitoring. Biosens. Bioelectron. 21:1405-1423.
Arduini, F. Ricci, F. Tuta, C.S. Moscone, D. Amine, A. and Palleschi, G.. 2006. Detection of carbamic and organophosphorous pesticides in water samples using a cholinesterase biosensor based on Prussian Blue-modified screen-printed electrode. Anal. Chim. Acta. 580: 155-162.
Artyukhin, A.B. Bakajin, O. Stroeve, P. and Noy, A. 2004. Layer-by-layer electrostatic self-assembly of polyelectrolyte nanoshells on individual carbon nanotube templates. Langmuir. 20: 1442-1448.
Bernabei, M. Chiavarii, S. Cremisini, C. and Palleschi, G. 1993. Anticholinesterase activity measurement by a choline biosensor: application in water analysis. Biosens. Bioelectron. 8: 265-271.
Caetano, J. and Machado, S.A.S. 2008. Determination of carbaryl in tomato �in natura� using an amperometric biosensor based on the inhibition of acetylcholinesterase activity. Sens. Actuators B. 129: 40-46.
Cesarino, I. Moraes, F.C. Lanza, M.R.V. and Machado, S.A.S. 2012. Electrochemical detection of carbamate pesticides in fruit and vegetables with a biosensor based on acetylcholinesterase immobilised on a composite of polyaniline-carbon nanotubes. Food Chem. 135: 873-879.
Chauhan, N. and Pundir, C.S. 2011. An amperometric biosensor based on acetylcholinesterase immobilized onto iron oxide nanoparticles/multi-walled carbon nanotubes modified gold electrode for measurement of organophosphorus insecticides. Anal. Chim. Acta. 701: 66-74.
Chen, X. Zhu, J. Xi, Q. and Yang, W.S. 2012. A high performance electrochemical sensor for acetaminophen based on single-walled carbon nanotube-graphene nanosheet hybrid films. Sens. Actuators B. 161: 648-654.
De Kok, A. Hiemstra, M. and Assoc, J. 1992. Optimization, automation, and validation of the solid-phase extraction cleanup and on-line liquid chromatographic determination of N-methylcarbamate pesticides in fruits and vegetables. Anal. Chem. 75: 1063-1072.
Du, D. Chen, S.Z. Cai, J. and Song, D.D. 2007. Comparison of drug sensitivity using acetylcholinesterase biosensor based on nanoparticles-chitosan sol-gel composite. J. Electroanal. Chem. 611: 60-66.
Du, D. Chen, S.Z. Cai, J. and Zhang, A.D. 2007. Immobilization of acetylcholinesterase on gold nanoparticles embedded in sol-gel film for amperometric detection of organophosphorous insecticide. Biosens. Bioelectron. 23: 130-134.
Du, D. Chen, S.Z. Cai, J. and Zhang, A.D. 2008. Electrochemical pesticide sensitivity test using acetylcholinesterase biosensor based on colloidal gold nanoparticle modified sol-gel interface. Talanta. 74: 766-772.
Du, D. Chen, S.Z. Song, D.D. Li, H.L. and Chen, X. 2008. Development of acetylcholinesterase biosensor based on CdTe quantum dots/gold nanoparticles modified chitosan microspheres interface. Biosens. Bioelectron. 24: 475-479.
Du, D. Chen, W.J. Zhang, W.Y. Liu, D.L. Li, H.B. and Lin, Y.H. 2010 Covalent coupling of organophosphorus hydrolase loaded quantum dots to carbon nanotube/Au nanocomposite for enhanced detection of methyl parathion. Biosens. Bioelectron. 25: 1370-1375.
Du, D. Ding, J.W. Cai, J. and Zhang, A.D. 2007. Determination of carbaryl pesticide using amperometric acetylcholinesterase sensor formed by electrochemically deposited chitosan. Colloids. Surf. B. 58: 145-150.
Du, D. Wang, M.H. Cai, J. Tao, Y. Tu, H.Y. and Zhang, A.D. 2008. Immobilization of acetylcholinesterase based on the controllable adsorption of carbon nanotubes onto an alkanethiol monolayer for carbaryl sensing. Analyst. 133: 1790-1795.
Du, D. Ye, X.X. Cai, J. Liu, J. and Zhang, A.D. 2010. Acetylcholinesterase biosensor design based on carbon nanotube-encapsulated polypyrrole and polyaniline copolymer for amperometric detection of organophosphates. Biosens. Bioelectron. 25: 2503-2508.
Egu�laz, M. Villalonga, R. Yanez-Sedeno, P. and Pingarron, J.M. 2011. Designing electrochemical interfaces with functionalized magnetic nanoparticles and wrapped carbon nanotubes as platforms for the construction of high-performance bienzyme biosensors. Anal. Chem. 83: 7807-7814.
Feng, Q.L. Liu, K.P. Fu, J.J. Zhang, Y.Z. Zheng, Z.X. Wang, C.M. Du, Y.L. and Ye, W.C. 2012. Direct electrochemistry of hemoglobin based on nano-composite film of gold nanopaticles and poly (diallyldimethylammonium chloride) functionalized graphene. Electrochim. Acta. 60: 304-308.
Geim, A.K. and Novoselov, K.S. 2007. The rise of graphene. Nat. Mater. 6: 183-191.
Guan, H.N. Zhang, F.L. Yu, J. and Chi, D.F. 2012. The novel acetylcholinesterase biosensors based on liposome bioreactors-chitosan nanocomposite film for detection of organophosphates pesticides. Food Res. Int. 49: 15-21.
Huang, J.D. Lin, Q. Zhang, X.M. He, X.R. Xing, X.R. Lian, W.J. Zuo, M.M. and Zhang, Q.Q. 2011. Electrochemical immunosensor based on polyaniline/poly (acrylic acid) and Au-hybrid graphene nanocomposite for sensitivity enhanced detection of salbutamol. Food Res. Int. 44: 92-97.
Huang, J.D. Xing, X.R. Zhang, X.M. He, X.R. Lin, Q. Lian, W.J. and Zhu, H. 2011. A molecularly imprinted electrochemical sensor based on multiwalled carbon nanotube-gold nanoparticle composites and chitosan for the detection of tyramine. Food Res. Int. 44: 276-281.
Iijima, S. 1991. Helical microtubules of graphitic carbon. Nature. 354: 56-58.
Kang, X.H. Wang, J. Wu, H. Aksay, I.A. Liu, J. and Lin, Y.H. 2009. Glucose oxidase-graphene-chitosan modified electrode for direct electrochemistry and glucose sensing. Biosens. Bioelectron. 25: 901-905.
Kim, B. Park, H. and Sigmund, W.M. 2003. Electrostatic interactions between shortened multiwall carbon nanotubes and polyelectrolytes. Langmuir. 19: 2525-2527.
Kim, B. and Sigmund, W.M. 2003. Self-alignment of shortened multiwall carbon nanotubes on polyelectrolyte layers. Langmuir. 19: 4848-4851.
Kok, F.N. and Hasirci, V. 2004. Determination of binary pesticide mixtures by an acetylcholinesterase-choline oxidase biosensor. Biosens. Bioelectron. 19: 661-665.
Li, Y. Yuan, R. Chai, Y.Q. and Song, Z.J. 2011. Electrodeposition of gold-platinum alloy nanoparticles on carbon nanotubes as electrochemical sensing interface for sensitive detection of tumor marker. Electrochim. Acta. 56: 6715-6721.
Liu, K.P. Zhang, J.J. Yang, G.H. Wang, C.M. and Zhu, J.J. 2010. Direct electrochemistry and electrocatalysis of hemoglobin based on poly (diallyldimethylammonium chloride) functionalized graphene sheets/room temperature ionic liquid composite film. Electrochem. Commun. 12: 402-405.
Ma, R.Z. Sasaki, T. and Bando, Y. 2004. Layer-by-layer assembled multilayer films of titanate nanotubes, Ag- or Au-loaded nanotubes, and nanotubes/nanosheets with polycations. J. Am. Chem. Soc. 126: 10382-10388.
Manso, J. Mena, M.L. Y��ez-Sede�o, P. and Pingarr�n, J.M. 2008. Alcohol dehydrogenase amperometric biosensor based on a colloidal gold-carbon nanotubes composite electrode. Electrochim. Acta. 53: 4007-4012.
McGarvey, B.D. 1993. High-performance liquid chromatographic methods for the determination of N-methylcarbamate pesticides in water, soil, plants and air. J. Chromatogr. 642: 89-105.
Miwa, D.W. Malpass, G.R.P. Machado, S.A.S. and Motheo, A.J. 2006. Electrochemical degradation of carbaryl on oxide electrodes. Water Res. 40: 3281-3289. 
Moraes, F.C. Mascaro, L.H. Machado, S.A.S. and Brett, C.M.A. 2009. Direct electrochemical determination of carbaryl using a multi-walled carbon nanotube/cobalt phthalocyanine modified electrode. Talanta. 79: 1406-1411.
Perez-Lopez, J.A. Zapardiel, A. Bermejo, E. Arauzo, E. and Hernandez, L. 1994. Electrochemical determination of carbaryl oxidation in natural water and soil samples. Fresenius J. Anal. Chem. 350: 620-625.
Pietrogrande, M.C. Blo, G. and Bighi, C. 1985. High-performance liquid chromatographic determination of naphthols as 4-aminoantipyrine derivatives : Application to carbaryl. J. Chromatogr. 349: 63-68.
Rong, L.Q. Yang, C. Qian, Q.Y. and Xia, X.H. 2007. Study of the nonenzymatic glucose sensor based on highly dispersed Pt nanoparticles supported on carbon nanotubes. Talanta. 72: 819-824.
Rouse, J.H. and Lillehei, P.T. 2003. Electrostatic assembly of polymer/single walled carbon nanotube multilayer films. Nano Lett. 3: 59-62.
Sattarahmady, N. Heli, H. and Moosavi-Movahedi, A.A. 2010 An electrochemical acetylcholine biosensor based on nanoshells of hollow nickel microspheres-carbon microparticles-Nafion nanocomposite. Biosens. Bioelectron. 25: 2329-2335.
Shan, C.S. Yang, H.F. Song, J.F. Han, D.X. Ivaska, A. and Niu, L. 2009. Direct electrochemistry of glucose oxidase and biosensing for glucose based on graphene. Anal. Chem. 81: 2378-2382.
Shan, D. Han, E. Xue, H.G. and Cosnier, S. 2007. Self-assembled films of hemoglobin/laponite/chitosan:application for the direct electrochemistry and catalysis to hydrogen peroxide. Biomacromolecules. 8: 3041-3046.
Shi, M. Xu, J.J. Zhang, S. Liu, B.H. and Kong, J.L. 2006. A mediator-free screen-printed amperometric biosensor for screening of organophosphorus pesticides with flow-injection analysis (FIA) system. Talanta. 68: 1089-1095.
Song, Y.H. Zhang, M. Wang, L. Wan, L.L. Xiao, X.P. Ye, S.H. and Wang, J.R. 2011. A novel biosensor based on acetylecholinesterase/prussian blue-chitosan modified electrode for detection of carbaryl pesticides. Electrochim. Acta. 56: 7267-7271.
Sotiropoulou, S. and Chaniotakis, N.A. 2005. Lowering the detection limit of the acetylcholinesterase biosensor using a nanoporous carbon matrix. Anal. Chim. Acta. 530: 199-204.
Sun, X. Du, S.Y. and Wang, X.Y. 2012. Amperometric immunosensor for carbofuran detection based on gold nanoparticles and PB-MWCNTs-CTS composite film. Eur. Food Res. Technol. 235: 469-477.
Sun, X. and Wang, X.Y. 2010. Acetylcholinesterase biosensor based on prussian blue-modified electrode for detecting organophosphorous pesticides. Biosens. Bioelectron. 25: 2611-2614.
Vakurov, A. Simpson, C.E. Daly, C.L. Gibson, T.D. and Millner, P.A. 2005. Acetylecholinesterase-based biosensor electrodes for organophosphate pesticide detection: II. Immobilization and stabilization of acetylecholinesterase. Biosens. Bioelectron. 20: 2324-2329.
Walcarius, A. and Kuhn, A. 2008. Ordered porous thin films in electrochemical analysis. Trend. Anal. Chem. 27. 593-603.
Wang, D.W. Li, F. Liu, M. Lu, G.Q. and Cheng, H.M. 2008. 3D periodic hierarchical porous graphitic carbon material for high rate electrochemical capacitive energy storage. Angew. Chem. Int. Ed. 47: 373-376.
Wang, K. Liu, Q. Dai, L. Yan, J.J. Ju, C. Qiu, B.J. and Wu, X.Y. 2011. A highly sensitive and rapid organoph)NT[\km|��������C	D	Y	Z	[	v	w	z	�	�	�����������ɹɹɦ��t[t[t[t[0h_zh�0zB*CJOJQJaJnHo(phtH-h_zh�0zB*CJOJQJaJnHphtHh�0zh�0zCJOJQJaJh�0zCJOJQJaJ$h�0zhkB*CJKHaJo(phh�0zB*CJKHaJo(phh�0zB*CJKHaJphh�0zh�f�5�CJaJo(h�0zhk5�CJaJo(h�0zhk5�CJaJ��D	�	���wm ��������tff
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�����������������������������������ڷ���ڷ����������h�x6h��o(	h�o(hS{�hS{�mHo(sHhS{�hS{�mHsHhS{�h�n�mHo(sHhS{�h�n�mHsHhS{�hS{�UhS{�	hS{�o(	h�n�o(h�n�Cosphate biosensor based on enhancement of CdS-decorated graphene nanocomposite. Anal. Chim. Acta. 695: 84-88.
Wang, Y. Li, Y.M. Tang, L.H. Lu, J. and Li, J.H. 2009. Application of graphene-modified electrode for selective detection of dopamine. Electrochem. Commun. 11: 889-892.
Wang, Y.Y. Wang, X.S. Wu, B.Y. Zhao, Z.X. Yin, F. Li, S. Qin, X. and Chen, Q. 2008. Dispersion of single-walled carbon nanotubes in poly (diallyldimethylammonium chloride) for preparation of a glucose biosensor. Sens. Actuators B. 130: 809-815.
Xiao, Y.H. and Li, C.M. 2008. Nanocomposites: from fabrications to electrochemical bioapplications. Electroanal. 20: 648-662.
Yang, T. Zhou, N. Zhang, Y.C. Zhang, W. Jiao, K. and Li, G.C. 2009. Synergistically improved sensitivity for the detection of specific DNA sequences using polyaniline nanofibers and multi-walled carbon nanotubes composites. Biosens. Bioelectron. 24: 2165-2170.
Yin, H.S. Ai, S.Y. Xu, J. Shi, W.J. and Zhu, L.S. 2009. Amperometric biosensor based on immobilized acetylcholinesterase on gold nanoparticles and silk fibroin modified platinum electrode for detection of methyl paraoxon, carbofuran and phoxim. J. Electroanal. Chem. 637: 21-27.
Zhang, H. Fan, L.Z. and Yang, S.H. 2006. Significantly accelerated direct electron-transfer kinetics of hemoglobin in a C60-MWCNT nanocomposite film. Chem. Eur. J. 12, 7161-7166.
Zhang, J. Lia, J. Yang, F. Zhang, B.L. and Yang, X.R. 2009. Preparation of Prussian blue@Pt nanoparticles/carbon nanotubes composite material for efficient determination of H2O2. Sens. Actuators B. 143: 373-380.
Zhang, R.Y. and Wang, X.M. 2007. One step synthesis of multiwalled carbon nanotube/gold nanocomposites for enhancing electrochemical response. Chem. Mater. 19: 976-978.
Zhao, W. Ge, P.Y. Xu, J.J. and Chen, H.Y. 2009. Selective detection of hypertoxic organophosphates pesticides via PDMS composite based acetylcholinesterase -inhibition biosensor. Environ. Sci. Technol. 43: 6724-6729.
Zhou, M. Guo, J.D. Guo, L.P. and Bai, J. 2008. Electrochemical sensing platform based on the highly ordered mesoporous carbon-fullerene system. Anal. Chem. 80: 4642-4650.












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