MWNTs/AChE新型生物传感器的制备及其在氧化乐果快速检测中的应用

关桦楠，迟德富*，宇 佳

(1.哈尔滨商业大学食品工程学院，黑龙江 哈尔滨 150076；2.东北林业大学生命科学学院，黑龙江 哈尔滨 150040)

MWNTs/AChE新型生物传感器的制备及其在氧化乐果快速检测中的应用

关桦楠1,2，迟德富2,*，宇 佳2

(1.哈尔滨商业大学食品工程学院，黑龙江 哈尔滨 150076；2.东北林业大学生命科学学院，黑龙江 哈尔滨 150040)

采用层层自组装技术制备(壳聚糖/多壁碳纳米管/乙酰胆碱酯酶)多层膜，用以构建新型有机磷农药残留检测电流型生物传感器。利用扫描电子显微镜获得多层酶膜的显微结构，利用电化学工作站对新型生物传感器的性能进行测量。结果表明：生物传感器的多层酶膜的最适制备层数为5层；以氧化乐果作为有机磷农药模型，在0.25～1.50μmol/L和1.75～10.00μmol/L的氧化乐果浓度区间内，响应电流与抑制率呈现良好的线性关系，最低检出限达到10nmol/L；新型生物传感器具有良好的重现性和稳定性。

乙酰胆碱酯酶；生物传感器；碳纳米管；氧化乐果

Over the last decade, biosensors based on acetylcholinesterase (AChE) enzyme have emerged as a promising technique for toxicity analysis, environmental monitoring, food quality control and military investigations[1]. 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 (chromatographic and coupled chromatographic spectrometric procedures) by simplifying or eliminating sample preparation, thus decreasing the analysis time and cost[2]. Most AChE biosensors reported to date are utilizing an electrochemical detection system. Fabrication of AChE biosensors involves immobilization of the AChE enzyme onto an electrode surface. Various immobilization strategies and materials have been used for this purpose including physical and covalent binding and affinity interactions[3].

Due to the poor adsorptive capacity of enzymes onto the electrode surface, the immobilization technique of enzyme onto the electrode is the key step in the construction of biosensor. Recently, a layer-by-layer (LBL) deposition technique for constructing multilayer films has attracted much attention because of its simplicity in procedure and wide choice of materials[4]. This technique involves analternate adsorption of enzyme and another electrolyte from solution onto an electrode surface through electrostatic force or covalently[5]. Combinations of different templates with synthetic and natural polyelectrolytes and nanoparticles can be applied leading to a pool of potential product[6].

Nano-materials have been used to improve the operational characteristics of enzyme-base biosensors, the improvement resulting from both the increased surface area and increased catalytic activity[7]. Carbon nanotubes (CNTs) are a new kind of carbon materials discovered by Iijima in 1991[8]. The carbon nanotubes are seamless nanometer size tubes curled formatted by monolayer or multilayer graphite flake[9]. Based on the different carbon atomic layer number of carbon nanotubes, the carbon nanotubes can be classified into two kinds[10]: the single-walled carbon nanotubes (SWNTs) and the multi-walled carbon nanotubes (MWNTs). Because of the special tube structure, the carbon nanotubes possess many unique properties such as good electrical conductivity, strong adsorptive ability, excellent bioconsistency and high strength which enable them to be used for the development of superconductive devices for microelectro-mechanical (MEM) and nanoelectromechanical (NEM) system applications[11]. Owing to these properties, especially the excellent conductive, adsorptive and bioconsistent properties, the carbon nanotubes are recently used even in the region of biosensor.

Taking account of the advantages of MWNTs and chitosan, the combination of MWNTs and chitosan will be very promising for constructing amperometric biosensors. In this work, we reported to prepare AChE biosensor using multilayers of MWNTs and AChE as electron intermediator through LBL technique, and on the design of the novel biosensor for the detection of organophosphate pesticides using dimethoate as example. The sensor will be further applied to the analysis of organophosphate pesticides in environmental monitoring.

1 Materials and Methods

1.1 Materials

Acetylcholinesterase (AChE), acetylthiocholine chloride (ATCl), phosphate buffered saline containing 0.1 mol/L KCl (PBS, 0.01 mol/L, pH 7.4), hydrochloric acid (37%), dithiobis (2-nitrobenzoicacid) (DTNB), bovine serum albumin (BSA) and chitosan were purchased form Sigma Aldrich. Enzyme solution was prepared in phosphate buffered saline (PBS, pH7.0) and its activity determined by Ellman’s

assay[12]. Dimethoate was the product of AccuStandard (USA). MWNTs (95% purity) were obtained from Institute of Chinese Academy of Science (China). All other reagents were of analytical grade and used without further purification. All aqueous solutions were prepared with doubly distilled water. PBS buffer was employed as supporting electrolyte. All experiments were performed in PBS at room temperature, approximately 25 ℃.

1.2 Preparation of MWNTs/AChE multilayer film coated Pt electrode via LBL

The multilayer films were assembled on the Pt electrode surface according to the reported procedures[13]. The Pt electrode was thoroughly polished using an alumina powder, etched for3 min in a 1:3:4 (V/V) mixture of HNO3:HCl:H2O, and then sonicated for4 min in distilled water.

The cleaned Pt electrode was immersed in polycation chitosan for 25 min and rinsed in PBS for5 min. The chitosan-coated Pt electrode was immersed in a MWNTs solution (1 mg/mL, pH 3.8, treated with acetic acid) for 15 min at room temperature to deposit the first monolayer of MWNTs on the electrode surface, and washed with the PBS for about5 min. The MWNTs-modified electrode was then immersed in polycation chitosan for 15 min and rinsed in PBS for5 min. The chitosan-MWNTs-modified electrode was then immersed in the AChE solution (100 U/mL, 0.1% BSA) for 20 min to immobilize AChE film, and also washed with the phosphate buffer solution for about5 min. These procedures were repeated up to10 times to construct a MWNTs/AChE multiplayer composed of10 alternate layers of MWNTs and AChE. In order to make the MWNTs being distributed on the electrode uniformly, the MWNTs solution was stirred by a magnetic chip. After then, the electrode was removed from phosphate buffer solution, rinsed gently with purified water, blotted dry around the edges and covered to protect from dust.

Surface images of substrate modified by (MWCNTS/ AChE)5were obtained by scanning electron microcopy (SEM, JEOL JSM-6460LV SEM, Japan) measurement operated at an accelerating voltage of 25.0 kV. The substrate was rinsed with doubly distilled water for10 min before SEM imaging in order to avoid the interference of salt crystallization from buffer solutions.

1.3 Electrochemical measurements

Electrochemical measurements were performed on a LK98II electrochemical workstation (Lanlike Instrument Company of Tianjin, China) with a conventional threeelectrode cell. A Pt electrode (Leici Electrode Companyof Shanghai, China) was used as the working electrode. An Ag/AgCl electrode and a platinum wire were used as the reference and counter electrodes, respectively. All the potentials in this paper were in respect to the Ag/AgCl reference electrode. Unless stated otherwise, electrochemical measurements were carried out in a phosphate buffer solution (PBS, 0.1 mmol/L, pH 7.0) at room temperature ((25±2)℃).

Cyclic voltammetric and amperometric measurements were used to characterize the (MWNTs/AChE)nbiosensor. For amperometric measurements, the electrochemical cell was stirred with a small magnetic bar at 150 r/min. After stabilization of the capacitive current, the enzymatic reaction was initiated by addition of3 mmol/L ATCl substrate and the response of the sensor was measured.

Inhibition measurements were carried out in a two steps batch procedure[2]by measuring the response of the sensor to additions of a constant amount of ATCl substrate (1 mmol/ L) before and after inhibition with pesticides. All inhibition measurements were carried out with dimethoate as a model organophosphate pesticide. The electrodes were incubated for10 min in doubly distilled water in the absence and presence of concentrations of dimethoate ranging from 10-8-10-5mol/L.

After being inhibited by dimethoate, (MWNTs/AChE)n/ Pt was washed with PBS and reactivated with 4.0 mmol/L pralidoxime iodide for8 min, then transferred to electrochemical cell of 1.0 mL pH 7.0 PBS containing 0.3 mmol/L ATCl to study the electrochemical response. The reactivation efficiency (R) was estimated as the method adopted earlier[3].

2 Results and Discussion

2.1 Effect of the number of MWNTs/AChE layers to the output current

Fig.1 shows the output current of the biosensors to 0.3 mmol/L ATCl, as a function of the number of MWNTs/AChE layers. The output current was enhanced with the increasing number of MWNTs/AChE layers and reached a maximum value at the5 layers, and then the output current was reduced on further increasing the number of number MWNTs/AChE layers. For one to five layers, the current increased with the increasing number of layers, which suggests the amounts of AChE increased with increasing the layers. The output current was enhanced in nearly linearity because each layer had almost the same quality of AChE. For 6-10 layers, on the other hand, the quality of AChE was tended to be saturation and the response was reduced.

Fig.1 Effect of the number of MWNTs/AChE layers (n) on the magnitude of the output current of the Pt-based AChE biosensor with 0.3 mmol/L ATCl in PBS (pH 7.0)

2.2 Characterization of MWNTs/AChE multilayer films

The typical SEM images of MWNTs (Fig.2A) and (MWNTs/AChE)5(Fig.2B) composite were presented in Fig. 2. It was clear that (MWNTs/AChE)5were in bundles with a disordered arrangement. When AChE was coated on the MWNTs, the obtained composite was much thicker than the raw MWNTs. However, the resultant composite possesses uniform coating and presents randomly oriented quasi-nanobelt like structures. The positively charged chitosan not only provided a reagent for dissolving MWNTs, but also provided a possibility for electrode to adsorb more negatively charged AChE, an opportunity to build up more AChE layers on Pt electrode, and a favorable microenvironment for AChE. AChE is used as a negatively charged material for preparing the multilayer films around pH 7.0, because the isoelectric point (pI) of AChE lies around pH 5.0[14], which can interact with chitosan by electrostatic adsorption. In this work, the physical separation of the individual layer in the multilayer films is assumed on the basis of the regular, step-wise mass increase during the assembly, however, the layer separation may not be definite, including overlapping and reconstructing between layers, so the structure of the resulting surface is unknown (but it is usually unknown with other procedures as well)[5,15].

Fig.2 SEM images of MWNTs (A) and the (MWNTs/AChE)5composite (B)

2.3 Electrochemical behavior of (MWNTs/AChE)5/Pt electrode

Cyclic voltammetry (CV) is a useful tool to evaluate the performance of (MWNTs/AChE)5composite films. The presence of (MWNTs/AChE)5, with their conductiveproperties and catalytic behavior promotes electrontransfer reactions at a certain potential.

Fig.3 Cyclic voltammograms of different electrodes.

As shown in Fig.3, no peak was observed when the bare Pt electrode (curve a) and (MWNTs/AChE)5/Pt electrode (curve b) were placed in pH 7.0 PBS. When 0.3 mmol/L ATCl was added to PBS, the CV response at the (MWNTs/AChE)5/ Pt electrode displayed an irreversible oxidation peak at 0.6 V (curve f). Obviously this peak arose from the oxidation of thiocholine, which was the hydrolysis product of ATCl and catalyzed by the immobilized AChE. This oxidation current was much higher than that at the AChE/Pt electrode (curve c) in pH 7.0 PBS containing 0.3 mmol/L ATCl and the peak potential shifted negatively about 50 mV comparing to AChE/Pt electrode (curve c). This was due to the presence of MWNTs in nanocomposite film, which possessed inherent conductive properties and catalytic behavior, thus it can provide a conductive pathway to electron transfer. The improvement in the response of the electrode could also be attributed to the increased the surface area in the presence of MWNTs to capture a large amount of enzymes, thus amplifying the current response.

2.4 Effect of dimethoate on response of (MWNTs/AChE)5/ Pt electrode

Dimethoate, one of an organophosphate pesticide, was selected as a model compound to study the inhibition effect on AChE. Upon construction (MWNTs/AChE)5/Pt was immersed in the standard solution of dimethoate at a known concentration (10-5mol/L and 10-7mol/L) for 15 min, the produced current decreased drastically (Fig.3, curve d and e). This was because dimethoate as one of the organophosphate pesticides exhibited fairly high acute toxicity and involved in the irreversible inhibition action on AChE, thus reduced the enzymatic activity to its substrate. Due to the notable change in voltammetric signal of the (MWNTs/AChE)5/Pt, a simple method for determination of dimethoate was established. With an increase concentration of dimethoate concentration, the peak current decreased. The peak current observed in the simple electronic voltammetric sensing system reflected the activity of immobilized enzyme, which could be used to detect trace organophosphate pesticides exposure. As shown in Fig.4, the inhibition efficiency of dimethoate was a linear function of its concentration from 0.25 to 1.5 μmol/L and 1.75 to 10.00 μmol/L. The linearization equation were I/% = 23.55c + 13.65% and I/% = 1.98c + 57.66%, with the correlation coefficients of 0.9981 and 0.9914, respectively. The detection limit was calculated to be10 nmol/L at a signal-to-noise ratio of 3, which was lower than other studies[16-17]. Furthermore, the inhibition rate of dimethoate decreased when increasing the concentration of dimethoate, indicating that the binding sites between pesticides and enzymes could reach saturation and equilibrium.

Fig.4 Relationship between peak currens and dimethoate concentration. Insets show the calibration curves for dimethoate determination.

2.5 Reactivation of the biosensor

A crucial problem for practical applications is the strong inhibition on AChE, which limits the reuse of biosensor. In order to settle this problem, reactivation of the enzyme by oximes has been investigated[18-19]. The (MWNTs/AChE)5/ Pt electrode inhibited by dimethoate can be completely reactivated when using nucleophilic compounds such as pralidoxime iodide. With increasing reactivation time, the reactivation ef ciency increased and reached a constant value after8 min. The inhibited AChE could be regenerated more than 93.6% of its original activity after immerging in 5.0 mmol/L pralidoxime iodide. Based on this reactivation procedure, the proposed biosensor can be used repeatedly with an acceptable reproducibility.

2.6 Precision of measurements and stability of biosensor

The intra-assay precision of the biosensors was evaluated by assaying one enzyme electrode for eight replicate determinations in 0.3 mmol/L ATCl after being immersedin the 3.0 μmol/L dimethoate solution for 8min. Similarly, the inter-assay precision, or fabrication reproducibility, was estimated at eight different electrodes. The RSD of intraassay and inter-assay were found to be 5.8% and 7.6%, respectively, indicating acceptable reproducibility. When the enzyme electrode was not in use, it was stored at4 ℃ in dry condition. No obvious decrease in the response of ATCl was observed in the rst 5-day storage. After a 20-day storage period, the sensor retained 85% of its initial current response, indicating acceptable stability of biosensor.

3 Conclusion

The AChE can catalyze the substance to hydrolyze selectively. And the activity of acetylcholinesterase can be restrained by the organophosphate and carbamate pesticides. We have successfully constructed a novel amperometric AChE biosensor based on the multilayer films composed of (MWNTs/AChE)5on the Pt electrode surface for highly selective and sensitive determination of organophosphate pesticides residues. The constructed copolymer network incorporating MWNTs provided a biocompatible microenvironment around the enzyme molecules to stabilize their biological activity and prevented them from leaking out of the interface. Due to the fast electron transfer of MWNTs, the resulting AChE biosensor exhibited high af nity to its substrate and produced a detectable and fast response. Based on the notable change in voltammetric signal, the simple method for screening of organophosphate pesticides exposure was established. The resulting AChE biosensor exhibited high sensitivity, good reproducibility, long-term stability and lowcost processes, which provided a new promising tool for the characterization of enzyme inhibitors and pesticide analysis. In this study, we make use of the characteristic to design the biosensors to detect the content of organophosphorus pesticides. We will propose a simple and efficient method for detection trace pesticide residues based on immobilization of AChE on nanosturctured materials modified electrode, which called electrochemical biosensor for screening of carbamate pesticides.

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TQ450.7

A

1002-6630(2013)04-0006-05

2011-10-06

国家自然科学基金青年科学基金项目(31201376)

关桦楠(1983—)，男，讲师，博士，研究方向为食品安全。E-mail：guanhuanan3@163.com

*通信作者：迟德富(1962—)，男，教授，博士，研究方向为森林保护学。E-mail：chidefu@126.com