Synthesis of highly dispersed zinc oxide nanoparticles on carboxylic graphene for development a sensitive acetylcholinesterase biosensor

Sensors and Actuators B 190 (2014) 730–736

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Synthesis of highly dispersed zinc oxide nanoparticles on carboxylic graphene for development a sensitive acetylcholinesterase biosensor Guangcan Wang a , Xincheng Tan b , Qing Zhou b,∗ , Yongjun Liu a , Min Wang a , Long Yang a a b

School of Chemical Science and engineering, Yunnan University, Kunming 650091, China School of Physical science and technology, Yunnan University, Kunming 650091, China

a r t i c l e

i n f o

Article history: Received 26 April 2013 Received in revised form 10 August 2013 Accepted 9 September 2013 Available online 17 September 2013 Keywords: ZnO nanoparticles Carboxylic graphene Nafion Acetylcholinesterase biosensor Pesticides

a b s t r a c t Highly dispersed zinc oxide nanoparticles (ZnO NPs) were synthesized on carboxylic graphene (CGR). A novel acetylcholinesterase (AChE) biosensor based on ZnO NPs, CGR and Nafion (NF) hybrids modified glass carbon electrode (GCE) has been successfully developed. ZnO NPs-CGR was homogeneously dispersed in NF and dropped on the surface of GCE. ZnO NPs-CGR-NF possessed excellent conductivity, catalysis and biocompatibility which were attributed to the synergistic effects of ZnO NPs, CGR and NF. ZnO NPs-CGR-NF/GCE provided a hydrophilic surface for AChE adhesion. The AChE biosensor showed favorable affinity to acetylthiocholine chloride (ATCl) and could catalyze the hydrolysis of ATCl with an apparent Michaelis-Menten constant value of 126 ␮M, which was then oxidized to produce a detectable and fast response. Under optimum conditions, the biosensor detected chlorpyrifos and carbofuran ranging from 1.0 × 10−13 to 1 × 10−8 M and from 1.0 × 10−12 to 1 × 10−8 M. The detection limits for chlorpyrifos and carbofuran were 5 × 10−14 M and 5.2 × 10−13 M, respectively. The developed biosensor exhibited many advantages such as good sensitivity, stability, reproducibility and low cost, thus providing a promising tool for analysis of enzyme inhibitors. This study could provide a universal platform for meeting the demand of the effective immobilization enzyme on the electrode surface. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Pesticides are widely used in agriculture due to their high efficiency as insecticides. Unfortunately, these compounds exhibit high acute toxicity, with the majority being hazardous to both human health and the environment. Indeed, the inhibition of AChE activity by pesticides can lead to a disturbance of normal neuronal function and possibly death [1,2]. Therefore the exact and speedy measurement of pesticides in water and food is of great importance. Biosensors based on AChE have emerged as a promising technique for toxicity analysis, environmental monitoring, food quality and military investigations in recent years [3,4]. The main application of AChE biosensors is for the detection of organophosphate and carbamate pesticides based on enzyme inhibition. These devices are designed to complement or replace the existing reference analytical methods such as HPLC, GC, GC/MS and etc. by simplifying or eliminating sample preparation, thus decreasing the analysis time and cost. Our research purpose is to develop a sensitive and stable AChE biosensor for detection of pesticides to reach the same level of these analytical instruments.

∗ Corresponding author at: 2 North Cuihu Road, Kunming 650091, PR China. Tel.: +86 871 65033774; fax: +86 871 65153832. E-mail address: [email protected] (Q. Zhou). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.09.042

Graphene, a two-dimensional sheet of sp2 -bonded carbon atoms arranged in a honeycomb lattice, has attracted increasing attention since it was first isolated from three-dimensional graphite by mechanical exfoliation [5]. Due to its extraordinary thermal, mechanical, and electrical properties, graphene is usually considered as a competitive candidate for next-generation electronic applications such as super-capacitors [6], batteries [7], sensors [8,9], biosensors [10,11], catalysts [12,13], etc. However, many researches have reported that the pure graphene actually exhibit unsatisfactory electrical conductivity because of the inevitable aggregation [14,15]. A useful method to prepare functionalized graphene is incorporated into chemical functional groups by covalent bonding on the graphene sheets. Some of the useful and unique properties of graphene can only be realized after it functioned with organic groups such as hydroxyl, carboxyl, amino and the like [14–16]. Nanostructured metal oxide semiconductors possess high surface area, nontoxicity, good biocompatibility, catalytic activity, chemical stability. They have been investigated for various applications such as solar cells, electrochemistry sensors and biosensor. Among the metal oxide semiconductors, ZnO NPs with the wurtzite crystal structure is an n-type semiconductor with a wide, direct band gap of 3.37 eV room temperature has been investigated for various applications such as photocatalysts [17,18], dye sensitized solar cells [19] and biosensor [20]. In recent years,

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the nanocomposite of ZnO NPs and graphene was synthesized and applied in the investigation fields of electrochemistry such as solar cells, photoluminescence, gas sensor, non-enzymatic hydrogen peroxide and glucose sensor, photocatalytic and antibacterial properties of graphene-ZnO NPs hybrids [21–24]. ZnO NPs were uniformly dispersed on functionalized graphene sheets and formed homogeneous ZnO NPs on functionalized graphene sheets. According to the results of investigation [14,15], we developed the AChE biosensor based on ZnO NPs-CGR-NF nanocomposites. Nafion (NF) polymer is chemically inert, ideal conductivity, hydrophilic, and insoluble in water, and thus possesses almost ideal properties for preparation of modified electrodes [25]. Some nanomaterials with high conductivity and catalytic activity are combined with NF and used to modify electrode that seems to be a possible approach to improve the sensitivity, selectivity and stability of the modified electrode. For example, Kumaravel et al. reported that nanosilver/NF electrode for electrochemical detection of methyl parathion showed strong electro catalytic activity, good stability and reproducibility [26]. Li et al. presented that NF-graphene nanocomposite film modified electrode for determination cadmium exhibited high sensitivity [27]. Li et al. proposed that horseradish peroxidase biosensor based on NF/graphene electrode for determination of H2 O2 exhibited both good operational storage and storage stability [28]. Chitosin (CS) is an abundant natural biopolymer with excellent film forming ability, biocompatibility and nontoxicity, which provides natural microenvironment to the enzyme and also gives sufficient accessibility to electrons to shuttle between the enzyme and the electrode [29]. Based on the above researches, the ZnO NPs-CGR nanocomposite was homogeneously dispersed in NF then dropped on the surface of GCE and formed uniform membrane. Combining the excellent characteristics of ZnO NPs, CGR and NF, a novel

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AChE biosensor based on ZnO NPs-CGR-NF/GCE was developed. The ZnO NPs-CGR-NF possessed excellent conductivity, catalytic activity and biocompatibility which were attributed to the synergistic effects of ZnO NPs, CGR and NF. Furthermore, ZnO NPs-CGR-NF/GCE provided hydrophilic surface for AChE adhesion. CS was used to immobilize AChE on the surface of ZnO NPsCGR-NF/GCE to keep the AChE activities and assist electrons to shuttle between the enzyme and-CSNS-NF/GCE. Finally, NF was used as a protective membrane of the AChE biosensors to improve the stability of the biosensor. The biosensor exhibited excellent affinity to its substrate and the catalytic effect on the hydrolysis of ATCl. The biosensor has been demonstrated as a device with high sensitivity, acceptable stability and reproducibility for the analysis of ATCl and pesticides. More importantly, this study provides a universal platform for meeting the demand of the effective immobilization enzyme on the ZnO NPs-CGR-NF/GCE surface. The process of preparation ZnO NPs-CGR nanocomposites (A) and fabrication of the biosensor (B) was showed in Scheme 1.

2. Experimental 2.1. Chemicals ATCl, AChE (Type C3389, 500 U/mg from electric eel), CS (85% deacetylation) and NF (5% in lower aliphatic alcohols and water) were purchased from Sigma-Aldrich (St. Louis, USA). Chlorpyrifos and carbofuran (99.99%) were obtained from AccuStandard (USA). Graphite powder was purchased from Sinopharm Chemical Reageat Company. (China). Bovine serum albumin (BSA) and (CH3 COO)2 Zn·6H2 O was obtained from Shanghai Chemical Reagent Co. Ltd. (China). All other reagents were of analytical grade. Aqueous solutions were prepared with deionized (DI) water (18 M cm).

Scheme 1. The process of preparation ZnO NPs-CGR nanocomposite (A) and fabrication of the biosensor (B).

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2.2. Preparation of CGR Graphite oxide prepared by Hummers’ method [30] was suspended in water and exfoliated through ultrasonication for 2 h to obtain graphene oxide (GO) solution. GO solution was centrifuged at 3000 rpm to remove unexfoliated graphite oxide. CGR was prepared by a chemical method. Briefly, GO aqueous suspension (5 ml) was diluted as to give a concentration of 2.0 mg/ml, and then sonicated for 1 h to give a clear solution. 1.2 g NaOH and 1.0 g chloroacetic acid (Cl-CH2 -COOH) were added to the suspension and sonicated for 3 h to convert the –OH groups to –COOH via conjugation of acetic acid moieties. Sequentially the suspension was separated by centrifuging at a speed of 15,000 rpm, washed with DI water for several cycles, dried in an oven at 60 ◦ C to gain CGR [31]. 2.3. Synthesis of highly dispersed ZnO NPs-CGR nanocomposites The ZnO NPs-CGR were prepared as follows: briefly, 2.0 mg CGR was suspended in 2.0 ml of 0.62 mM (CH3 COO)2 Zn·6H2 O by sonicating for 10 min to disperse CGR equably. Then 1.0 ml of 0.01 M sodium citrate and 10.0 ml of ethanol were added to the above suspension. Ice-cold, freshly prepared 1.0 ml of 0.01 M NaBH4 solution was added to the above mixture while stirring until the color of the solution did not change. After stirring for an additional 10 h, the suspension was separated by centrifuging at a speed of 12,000 rpm, washed with DI water for several cycles, dried in an oven at 60 ◦ C. Then, Zn NPs were oxidized to ZnO NPs by heating at 120 ◦ C in atmosphere. To improve the crystallinity of ZnO NPs in CGR, the product was annealed at 300 ◦ C for 2 h. 2.4. Preparation of biosensors NF solution (0.125%, Wt/V) was prepared by diluting 5% of NF with ethanol and DI water (V/V, 1/1). The ZnO NPs-CGR (0.5 mg) were added to 1.0 ml of the NF solution and sonicated thoroughly until a homogeneous suspension of ZnO NPs-CGR-NF was obtained. Similarly 0.5 mg/ml CGR-NF and GO-NF homogeneous suspension was obtained. The suspensions were stored at 4 ◦ C. A GCE was polished carefully to a mirror-like with 0.3 and 0.05 ␮m alumina slurry and sequentially sonicated for 3-min in nitric acid (V/V, 1/1), ethanol and water. Before the experiment, the electrode was scanned from −0.1 to +1.1 V until a steady-state current–voltage curve was obtained. The ZnO NPs-CGR-NF/GCE was prepared by costing 5 ␮l of the 0.5 mg/ml ZnO NPs-CGR-NF suspension onto the GCE and drying at room temperature. A similar method was used to prepare CGR-NF/GCE and GO-NF/GCE. The enzyme solution was mixed as 0.05 U AChE and 0.2% of CS (Wt/V, 50 mM acetic acids). The modified electrodes were each coated 4.5 ␮l of AChE-CS (V/V, 2/1) and dried at 4 ◦ C. The AChE-CS/GO-NF/GCE, AChE-CS/CGRNF/GCE and AChE-CS/ZnO-CGR/GCE biosensors were obtained and washed with 0.1 M PBS to remove the unbound AChE. Finally, three types of biosensor were each covered with 3 ␮l 0.1% (Wt/V) NF as the protective membrane. Thus, three types of biosensors NF/AChE-CS/GO-NF/GCE, NF/AChE-CS/CGR-NF/GCE and NF/AChECS/ZnO-CGR/GCE were gained. Similarly, NF/AChE-CS/GCE was produced as a control.

(XRD, Rigaku TTRIII, Japan) was used to identify the phase of ZnO NPs on CGR nanosheets. 2.6. Measurements Electrochemical analysis of the biosensor was performed using an IM6ex (Zahner Elektrik Instruments, Germany) electrochemical work station. A conventional three-electrode system was employed with a saturated calomel electrode (SCE) as the reference electrode, platinum foil as the counter electrode, and the modified GCE (3 mm in diameter) as the working electrodes. Cyclic voltammetry (CV) measurements were performed in 0.1 M phosphate buffer solution (PBS, pH 7.4) between 0.0 and 1.0 V for characteristic investigations of NF/AChE-CS/ZnO NPsCGR-NF/GCE biosensors. The typical current-time CV plot for the biosensor was gained at 0.47 V after the successive addition of ATCl to PBS with stirring. The apparent Michaelis-Menten constant app (Km ) of the biosensor was calculated from the Line weaver-Burk equation: 1 = iss



app

Km imax



·

1 1 + C imax

(1)

where iss is the steady-state current after the addition of substrate, imax is the maximum current measured under saturated substrate app condition and C is the concentration of the substrate. The Km value, which gives an indication of the enzyme substrate kinetics for the biosensor, was determined by analysis of the slope and intercept of the plot of the reciprocals of steady-state current versus ATCl concentration. The obtained NF/AChE-CS/ZnO-CGR-NF/GCE was first immersed in 0.1 M pH 7.4 PBS containing different concentrations of standard pesticide at room temperature (25 ± 1 ◦ C) for 6-min and then transferred to the electrochemical cell of pH 7.4 PBS containing 0.5 mM ATCl to study the amperometric response by differential pulse voltammetry (DPV) between 0.20 and 0.75 V. The inhibition of pesticide was calculated as follows: inhibition(%) =

iP,control − iP,exp × 100% iP,control

(2)

where iP,control is the peak current of ATCl on NF/AChE-CS/ZnOCGR/GCE, iP , exp is the amperometric response of ATCl on NF/AChE-CS/ZnO-CGR/GCE with pesticide inhibition. The detection limit (LD) was calculated by using the equation given below [32]: LD =

3S b

(3)

where S is the standard deviation of the blank solution, b is the slope of the analytical curve. 2.7. Optimization of incubation time Inhibition of chlorpyrifos and carbofuran were tested by CV in terms of their effect on AChE activity at different incubation times (from 2 to 16 min) in a pesticide solution (10−10 M), respectively. 2.8. Interference study

2.5. Materials characterization Scanning electron microscopy (SEM, QUNT200 USA) was used to characterize CGR and ZnO-CGR morphologies. Raman spectra (Raman Station 400F PERKINELMER USA) and Fourier transform infrared spectrometry (FTIR, Thermo Fisher SCIENTIFIC Nicolet IS10 USA) were used to study the GO and CGR. X-ray diffractometer

The interfering species of glucose (0.5 mM), citric acid (0.5 mM), oxalic acid (0.5 mM), 0.5 mM p-nitrophenol, 0.5 mM nitrobenzene, 0.5 mM p-nitroaniline, 0.5 mM trinitrotoluene, 0.5 mM toluene and 0.5 mM p-toluenesulfonic acid were studied. The signal for 0.5 mM ATCl was compared with the signal obtained in the presence of the interfering species after incubated with 10−10 M chlorpyrifos.

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2.9. Precision of measurements and stability studies The intra-assay precision of the biosensor was evaluated by testing one NF/AChE-CS/ZnO-CGR -NF/GCE for six times in 0.5 mM ATCl after being immersed in the 1.0 × 10−10 M chlorpyrifos for 6-min. The inter-assay precision was estimated with six different biosensors in the same way. The RSD of intra-assay and inter-assay were demonstrated reproducibility of the biosensor. The stability of the biosensor was evaluated by testing the amperometric response of the NF/AChE-CS/ZnO-CGR -NF/GCE biosensor in 0.1 M PBS containing 0.5 mM ATCl by CV every five days. The retained ratio of its initial current response was indicated the stability of biosensor. 2.10. Preparation and determination of real samples Two samples, tap water sample and water sample from a natural lake, were filtered through a 0.22 ␮m membrane and the pH was adjusted to 7.4. After simple pretreatment, different concentrations of chlorpyrifos and carbofuran were added to study the recovery under the optimal conditions. 3. Results and discussion 3.1. Characterization of ZnO NPs-CGR Fig. 1 shows the SEM images of CGR and ZnO-CGR hybrids. The SEM image of Fig. 1a show a few layers crumpled sheets of CGR morphology with a dimension ranging from several hundred nm to several ␮m and 3 to 5 nm in thickness. Fig. 2b indicats ZnO NPs were coated on the surfaces of CGR sheets with well-separated. Raman spectra of graphite, GO and CGR are shown in Fig. 2a, respectively. Highly ordered graphite had only a couple of Ramanactive bands visible in the spectra, the in-phase vibration of the graphite lattice (G band) at 1576 cm−1 as well as the (weak) disorder band caused by the graphite edges (D band) at approximately 1355 cm−1 . Raman-active bands visible in the spectra of GO as universal are observed that higher disorder in graphite led to a broader G band, as well as to a broad D band of higher relative intensity compared to that of the G band. The G band broadens of GO significantly and displayed a shift to higher frequencies 1357 cm−1 and 1601 cm−1 (blue-shift), and the D band grows in intensity. Ramanactive bands visible in the spectra of CGR show the G band shifts back to the position of the G band in graphite, which is attributed to a graphitic “self-healing” similar to what is observed from the sharpening of the G peak and the intensity decrease of the D peak in heat-treated graphite [33,34]. In Fig. 2b, the IR spectra of CGR show the presence of –OH (3414 cm−1 ), C O (1733 cm−1 ) on CGR and confirm the presence of the carboxylic group. But on grapheme, IR data show the disappearance of the C O bands (at 1733 cm−1 ). XRD patterns of CGR and ZnO NPs-CGR are shown in Fig. 2c, respectively. All peaks of the ZnO NPs-CGR diffraction spectra are indexed as ZnO wurtzite structures which agree well with the values on the standard card (JCPDS Card No. 36-1451). No other characteristic peaks of the crystalline impurities were observed. ZnO NPs-CGR diffraction spectra indicated ZnO NPs were coated on the surfaces of CGR sheets. Furthermore, the characteristic diffraction peak of the CGR confirmed the presence of CGR in the ZnO NPs-CGR nanocomposites. 3.2. Optimization of the preparation of the biosensor Optimization of the ratio of ZnO NPs to ZnO NPs-CGR, the volume of ZnO NPs-CGR-NF, the concentration of Nafion, amount of AChE, biocompatibility of materials for AChE immobilization, stability

Fig. 1. SEM image of CGR (a) and ZnO NPs-CGR (b).

of biosensor, pH and temperature are supplied in Supplementary Materials. 3.3. Electrochemical behavior of the biosensors Under the optimal conditions, CVs of enzymatic product thiocholine produced by ATCl was investigated on NF/AChE-CS/NF/GCE, NF/AChE-CS/GO-NF/GCE, NF/AChE-CS/CGR-NF/GCE and NF/AChECS/ZnO NPs-CGR-NF/GCE biosensors as shown in Fig. 3. No amperometric response can be observed at the four biosensors (Curve a–d) in PBS (pH 7.4). However, when 0.5 mM ATCl was added into the PBS (pH 7.4), an obvious amperometric response was observed at the four biosensors (Curve e to h). Obviously, those amperometric responses were attributed to the oxidation of thiocholine, hydrolysis product of ATCl, which were catalyzed by immobilized AChE. Fig. 3 shows the oxidation peak currents increased and the oxidation peak potentials shifted negatively in sequence. At NF/AChE-CS/ZnO NPs-CGR-NF/GCE, the oxidation peak current is the highest and peak potential is the lowest of four biosensors (Curve h). Thus, the results indicate that ZnO NPs-CGRNF improve the conductivity and catalytic activity of the biosensor. The decrease of the overpotential of thiocholine oxidation is beneficial for avoiding interference from other electroactive species in biological mixtures. ZnO is biocompatible with a high isoelectric point (IEP) of about 9.5 [35], which make it suitable for absorption of low IEP

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Fig. 3. CV of NF/AChE-CS/GCE (a), NF/AChE-CS/GO-NF/GCE (b), NF/AChE-CS/SGRNF/GCE (c) and NF/AChE-CS/ZnO-CGR-NF/GCE (d) in 0.1 M PBS and in 0.1 M PBS containing 0.5 mM ATCl (e to h), Scan rate: 0.10 V/s in 25 ◦ C.

membrane of the AChE biosensors to to prevent the loss of the enzyme molecules, improve the anti-interference ability of the biosensor and provide a biocompatible microenvironment to maintain enzymatic activity. In Fig. 3 (curves from e to h), the oxidation peak currents increased orderly that indicated the conductivity of biosensors was improved. The oxidation peak potentials shifted to lower potentials orderly that indicated the catalysis of biosensors was enhanced. The high performance of the biosensor is due to the synergy of ZnO, graphene, chitosin and Nafion. ZnO-CGR-NF/GCE provides a simple platform of hydrophile, biocompatibility, high catalytic activity and conductivity for meeting the demand of the effective immobilization enzyme on the electrode surface. 3.4. Detection of ATCl

Fig. 2. (a) The Raman spectra of Graphite, GO and CGR; (b) The FTIR spectra of GR and CGR; (c) XRD of CGR and ZnO NPs-CGR.

proteins such as AChE (IEP = 4.5 [36]). Large surface area of ZnO nanoparticles can immobilize higher amount of enzyme molecules and provide direct electron transfer between the active sites of enzyme and electrode [35]. Therefore, ZnO can improve catalytic activity of CGR. Carboxylic graphene (CGR) with excellent conductivity, catalysis and high surface area [25] provided a platform to construct CGR-ZnO-NF membrance for preparation of modified electrodes. Chitosin was used to immobilize AChE on the surface of ZnO-CGR-NF/GCE to keep the AChE activities and assist electrons to shuttle between the enzyme and ZnO-CGR-NF/GCE. NF improves conductivity of ZnO-CGR and forms ZnO-CGR-NF membrane on the surface of GCE. Besides, NF was also used as a protective

Electrochemistry detection of ATCl was carried out between NF/AChE-CS/ZnO-CGR-NF/GCE and ATCl. Fig. S4 shows the CVs of the biosensor in 0.1 M pH 7.4 PBS at 0.47 V in a stirred solution, which is added an ATCl stock solution. With the increase of ATCl concentration, the amperometric response of the biosensor increased. The amperometric responses of the biosensor are a linear function of ATCl concentration in two segments: one is in low concertration from 0.2 ␮M to 30 ␮M; another is in high concertration from 30 ␮M to 500 ␮M. The detection limit was 0.1 ␮M. At higher ATCl concentrations the shape of the amperometric response is indicative of a Michaelis–Menten process (Fig. app S5). The Km in the present studies was calculated to be 126 ␮M according to Lineweaver-Burk equation. This value was lower than that for AChE adsorbed on reduced graphene oxide-gold nanocomposites modified electrode (0.16 mM) [37], for AChE immobilized on CdS-decorated graphene nanocomposite modified electrode (0.24 mM) [38] and for AChE adsorbed on liposome bioreactorschitosan nanocomposite film modified electrode (0.36 mM) [39] indicating that the AChE biosensor had a great affinity and catalysis to its substrate ATCl. 3.5. Effect of incubation time The activity of AChE was influenced by the time for which AChE was incubated with 10−10 M chlorpyrifos and carbofuran, respectively. The inhibition level of AChE increased with increasing incubation time (Fig. S6). Considering the relations of analytical time with sensitivity and stability of the amperometric measurements, an exposure time of 6 min was chosen as the best compromise between the signal and exposure time.

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ATCl was compared with the signal obtained in the presence of the interfering species. The test result showed no noticeable changes in the presence of 0.5 mM glucose, 0.5 mM citric acid and 0.5 mM oxalic acid respectively at the present operating potential in this system. However, the amperometric response decreased obviously in the presence of 0.5 mM p-nitrophenol, 0.5 mM nitrobenzene, 0.5 mM p-nitroaniline and 10−10 M carbofuran after incubated with 10−10 M chlorpyrifos. The results of the interference study are shown in Fig. S7, which indicate that p-nitrophenol, nitrobenzene, p-nitroaniline and carbofuran severely interfere the determination of chlorpyrifos.

3.8. Precision of measurements and stability of biosensor

Fig. 4. DPV of the NF/AChE-CS/ZnO-CGR-NF/GCE in 0.1 M PBS containing 0.5 mM ATCl after incubation with 0 (a), 10−13 M (b), 10−12 M (c), 10−11 M (d), 10−10 M (e), 10−9 M (f) and 10−8 M (g) chlorpyrifos for 6 min.

3.6. Detection of pesticides The detection limit and the linear working range of the biosensors were evaluated for the different pesticides. The responses of NF/AChE-CS/ZnO-CGR-NF/GCE biosensor to 0.5 mM ATCl were measured by DPV after incubation at different concentrations of chlorpyrifos and carbofuran, respectively. As shown in Fig. 4, the response of the biosensor before and after 6 min incubation in 10−13 , 10−12 , 10−11 , 10−10 , 10−9 and 10−8 M chlorpyrifos, the peak currents (curves b–g) dramatically decrease compared with that on the control (curve a), and the decrease in peak current increase with the increasing concentration of chlorpyrifos. Calibration plots of inhibition percentage versus pesticide concentration are shown in Fig. 5. Linear relationships between the inhibition percentage and the concentration of pesticides are obtained and were shown in Table S1. The two linear ranges indicate that the biosensor is more sensitive for detecting low concentration of pesticides than high concentration. The performances of the biosensor are compared with those of other AChE biosensors reported literatures [37,40–42] (Table S2). 3.7. Interference study The interfering signal due to the most common electroactive species was investigated. The signal for a fixed concentration of

The intra-assay precision of the biosensor was evaluated by assaying one enzyme electrode for six replicate determinations in 0.5 mM ATCl after being immersed in the 1.0 × 10−10 M chlorpyrifos for 6-min. Similarly, the inter-assay precision, or fabrication reproducibility, was estimated at six different electrodes. The RSDs of intra-assay and inter-assay were found to be 3.7% and 5.9%, respectively, indicating an acceptable reproducibility. When the enzyme electrode was not in use, it was stored at 4 ◦ C in dry condition. No obvious decrease in the response of ATCl was observed in the first 10-day storage. After a 30-day storage period, the sensor retained 89% of its initial current response, indicating the acceptable stability of biosensor.

3.9. Analytical real samples To further demonstrate the practicality of the proposed method, the recovery test was studied by the standard addition method. Table S3 shows the results obtained by analysis of these real samples. The recoveries of tap water and lake water were observed in the range of 93.2–104.8%, which demonstrated low matrix effect on the amperometric response. The low relative standard deviations for chlorpyrifos and carbofuran demonstrated the high precision of analysis.

4. Conclusion In this work, combining the advantageous characteristics of ZnO NPs and CGR, NF and CS, a novel AChE biosensor based on ZnO NPsCGR-NF has been developed. The ZnO NPs-CGR-NF with excellent conductivity, catalysis and biocompatibility offered an extremely hydrophilic surface for AChE adhesion. CS was used to immobilize enzymes on the surface of ZnO NPs-CGR-NF/GCE, keep biological activity of AChE and assist electrons to shuttle between AChE and the electrode modified. Finally, NF was used as a protective membrane of the biosensors to improve the stability of the biosensor. The biosensor exhibited many advantages such as low applied potential, fast response, high sensitivity, acceptable stability, reproducibility and simple fabrication. The biosensor has potential application in biomonitoring of chlorpyrifos and carbofuran pesticides and other organophosphate and carbamate pesticides. The method not only can be used to immobilize other enzymes to construct a range of biosensors but also may be extended to assemble other biological molecules, such as antibody, antigen and DNA for wide bioassay applications.

Appendix A. Supplementary data Fig. 5. Inhibition curves of NF/AChE-CS/ZnO-CGR-NF/GCE biosensor for chlorpyrifos and carbofuran determination by DPV.

Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2013.09.042.

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Biographies Guangcan Wang is an associate professor in the Department of Chemistry of Yunnan University, China. He received his PhD degree in Physical Electronics from Electronic research institute of Ministry of Information Industry Beijing in 2003. Dr. Wang’s research interests involve nanomaterial biosensors, physical and chemic sensor. Xincheng Tan is currently pursuing his Master’s degree in Yunnan University, China. His research interest is mainly focused on biosensor. Qing Zhou is a Professor in the Department of physics of Yunnan University, China. She received her PhD degree in Physical Electronics and Photoelectronics from University of Electronic Science and Technology of China in 2000. Prof. Zhou’s research interests involve nanomaterials and their application in biosensors and solar cells. Yongjun Liu received his Master’s degree from Yunnan University in 2001. He is an engineer in Center of Modern Analysis and Test, Yunnan University, China. His research interests cover SEM imaging of biomaterials and polymers and drug release system. Min Wang is an engineer in Center of Modern Analysis and Test, Yunnan University, China. Her research interests cover XRD and FTIR analysis. Long Yang is currently pursuing his Master’s degree in Yunnan University, China. His research interest is mainly focused on electrochemical biosensors.