A novel acetylcholinesterase biosensor for pesticide detection -

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A novel acetylcholinesterase biosensor for pesticide detection

A novel acetylcholinesterase (AChE) biosensor based on AgNPs, carboxylic graphene (CGR) and Nafion (NF) hybrid modified glass carbon electrode (GCE) has been successfully developed. AgNPs–CGR–NF possessed predominant conductivity, catalysis and biocompatibility and provided a hydrophilic surface for AChE adhesion. Chitosan (CS) was used to immobilize AChE on the surface of Ag NPs–CGR–NF/GCE to keep the AChE activities. 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 133 μM, which was then oxidized to produce a detectable and fast response. Under optimum conditions, the biosensor detected chlorpyrifos and carbaryl at concentrations 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 carbaryl were 5.3 × 10−14 M and 5.45 × 10−13 M, respectively. This study could provide a simple and effective immobilization platform for meeting the demand of the effective immobilization enzyme on the electrode surface.

Preparation of CGR

Graphite oxide prepared by Hummers’ method 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 in a vacuum freeze-drying. Briefly, GO aqueous suspension (5 ml) was diluted to give a concentration of 2.0 mg/ml, and then sonicated for 1 h to give a clear solution. 1.2 g of 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. After vacuum freeze-drying, CGR was obtained.

Synthesis of Ag NPs–CGR nanocomposites

The Ag NPs–CGR nanocomposites were prepared as follows: briefly, 2.0 mg CGR was suspended in 2.0 ml of 0.46 mM AgNO3 by sonicating for 10 min to disperse CGR equably. Then 1.0 ml of 0.01 M sodium citrate and 4.0 ml 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. After vacuum freeze-drying, Ag NPs–CGR nanocomposites were obtained.

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 Ag NPs–CGR (0.5 mg) were added to 1.0 ml of the NF solution and sonicated thoroughly until a homogeneous suspension of Ag NPs–CGR–NF obtained. Similarly 0.5 mg/ml CGR–NF and GO–NF homogeneous suspension was obtained, respectively. The suspensions were stored under refrigeration at 4 °C. A GCE was polished carefully to a mirror-like with 0.3 and 0.05 μm 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 Ag NPs–CGR–NF/GCE was prepared by dropping 5 μl of 0.5 mg/ml Ag NPs–CGR–NF suspension onto the surface of 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 with 0.05 U AChE and 0.2% CS (Wt/V, 50 mM acetic acids). The modified electrodes were each coated 4.5 μl of AChE–CS (V/V, 2/1) and dried at 4 °C. The AChE–CS/GO–NF/GCE, AChE–CS/CGR–NF/GCE and AChE–CS/Ag NPs–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 μl 0.1% (Wt/V) NF as the protective membrane. Thus, three types of biosensor structure were NF/AChE–CS/GO–NF/GCE, NF/ AChE–CS/CGR–NF/GCE and NF/AChE–CS/Ag NPs–CGR/GCE. Similarly, NF/AChE–CS/GCE was produced as a control.

Material characterization

Scanning electron microscopy, scanning probe microscopy and transmission electron microscopy were used to characterize CGR and Ag NPs–CGR morphologies. Raman spectra and Fourier transform infrared spectra were used to study the GO and CGR. An X-ray diffractometer was used to identify the phase of Ag NPs on CGR sheets. The solution CGR and Ag NPs–CGR 0.5 mg/ml of DI water respectively were used for SEM, AFM and TEM detection. GO and CGR which were not treated were used for Raman spectra detection. GO and CGR which were mixed with a fix quantity of potassium bromide were used for Fourier transform infrared spectrometry detection.

Measurements

Electrochemical analysis of the bioelectrodes was performed using an IM6ex 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 (diameter = 3 mm) 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/Ag NPs–CGR– NF/GCE biosensors. The apparent Michaelis–Menten constant of the biosensor was calculated from the Lineweaver–Burk equation.

Michaelis–Menten constant, which gives an indication of the enzyme substrate kinetics for the biosensor, 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/Ag NPs–CGR–NF/GCE was first immersed in 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 (DPV) between 0.2 and 0.75 V. The inhibition of pesticide was calculated.

Precision of measurements and stability studies

The intra-assay precision of the biosensor was evaluated by testing one NF/AChE–CS/Ag NPs–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 intra-assay and inter-assay RSDs demonstrated reproducibility of the biosensor. Stability was evaluated by testing the amperometric response of the NF/AChE–CS/Ag NPs–CGRNF/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 indicated the stability of biosensor.

Preparation and determination of real samples

Two samples, tap water sample and lake water sample, were filtered through a 0.22 μm membrane and the pH was adjusted to 7.4. After simple pretreatment, different concentrations of chlorpyrifos and carbaryl were added to study the recovery under the optimal conditions.

Results and discussion

Characterization of Ag NPs–CGR

The SEM image indicated a few layers of crumpled sheets of CGR morphology with a dimension ranging from several hundred nm to several μm and thickness of 1.1 nm. TEM indicated that Ag NPs were coated on the surfaces of CGR sheets well separated. Highly ordered graphite had only a couple of Raman-active 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. GO of Raman-active bands visible in the spectra as universal 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 GO significantly and displayed a shift to higher frequencies of 1357 cm−1 and 1601 cm−1 (blue-shift), and the D band grew in intensity. CGR of Raman-active bands visible in the spectra shows that the G band shifts back to the position of the G band in graphite, which was attributed to a graphitic “self-healing” similar to what was observed from the sharpening of the G peak and the intensity decrease of the D peak in heat-treated graphite. The appearance of strong peaks at 3404 cm−1 and 1733 cm−1 on CGR confirmed the presence of the carboxylic group. Peaks of the Ag NPs–CGR diffraction spectra could be indicated as Ag NPs which agreed well with the values on the standard card (JCPDS Card No. 04-0783). Ag NPs–CGR diffraction spectra indicated that Ag NPs were coated on the surfaces of CGR sheets.

Electrochemical behavior of NF/AChE–CS/Ag NPs–CGR–NF/GCE

When 0.5 mM ATCl was added into the PBS (pH 7.4), an obvious amperometric response was observed at NF/AChE–CS/GCE, NF/ AChE–CS/GO–NF/GCE, NF/AChE–CS/CGR–NF/GCE and NF/AChE–CS/Ag NPs–CGR–NF/GCE. The oxidation peak potentials shifted to lower potentials orderly that indicated the enhanced catalysis. Obviously, Ag NPs–CGR– NF with excellent conductivity and catalytic activity provided an extremely hydrophilic surface for AChE adhesion. The Ag NPs–CGR–NF possessed excellent conductivity, catalytic activity and biocompatibility which were attributed to the synergistic effects of Ag NPs, CGR and NF. CS was used to immobilize enzymes on the surface of Ag NPs–CSNS–NF/ GCE, keep the enzyme activities and improve electrons to shuttle between the enzyme and the electrode. Besides, NF protective membrane was used to prevent the loss of the enzyme molecules, improve the antiinterference ability of the biosensor and provide a biocompatible microenvironment to maintain enzymatic activity.

Detection of ATCl

Under optimal conditions, CVs were used to investigate the electrochemistry reaction between AChE and ATCl. With increasing ATCl concentration, the amperometric response of the biosensor increases. The amperometric response of the biosensor was a linear function of ATCl concentration in two segments. One was from 1 μM to 50 μM and the other was from 50 μM to 500 μM. The detection limit was 0.5 μM. The Km app in the present studies was calculated to be 133 μM according to the Lineweaver–Burk equation. This value was lower than that for AChE adsorbed on reduced graphene oxide–gold nanocomposite modified electrode (0.16 mM), for AChE immobilized on CdS-decorated graphene nanocomposite modified electrode (0.24 mM) and for AChE adsorbed on liposome bioreactors–chitosan nanocomposite film modified electrode (0.36 mM) indicating that the NF/AChE–CS/ Ag NPs–CGR–NF/GCE biosensor had a great affinity and catalysis to its substrate ATCl.

Effect of incubation time

Inhibition of chlorpyrifos and carbaryl were tested by CV in terms of their effect on AChE activity at different incubation times (2 to 16 min) in a pesticide solution (10−10 M), respectively. The inhibition level of AChE increased with increasing incubation time. Considering the relations of analytical time with sensitivity and stability of the amperometric measurements, an exposure time of min was chosen as the best compromise between the signal and exposure time

Pesticide detection

The inhibition effects of different pesticides were investigated by DPV measuring the response of the biosensor to 0.5 mM ATCl after incubation by different concentrations of chlorpyrifos and carbaryl, respectively. With the response of the biosensor before and after 6 min of incubation in 10−13, 10−12, 10−11, 10−10, 10−9 and 10−8 M chlorpyrifos, the peak currents (curves b–g) dramatically decreased compared with that on the control (curve a), and the decrease in peak current increased with the increasing concentration of chlorpyrifos. The two linear ranges of chlorpyrifos and carbaryl indicated that the biosensor was more sensitive for detecting low concentration of pesticides than high concentration of pesticides.

Interference study

The signal for a fixed concentration of ATCl was compared with the signal obtained in the presence of the interfering species after the biosensor was incubated in 10−10 M chlorpyrifos for 6 min. The test result shows that no noticeable changes of amperometric response were detected 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, 0.5 mM trinitrotoluene, 0.5 mM toluene, 0.5 mM p-toluenesulfonic acid and 10−10 M carbaryl.

Precision of measurements and stability of biosensor

The intra-assay precision of the biosensors was evaluated by assaying one enzyme electrode for six replicate determinations in 0.5 mM ATCl after being immersed in 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 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 88% of its initial current response, indicating the acceptable stability of biosensor.

To investigate the accuracy of an analytical method, spike recovery is a useful tool. The variability was low if there were no interferences or matrix effects so that recovery close to 100% was expected. A standard addition method was adopted to estimate the accuracy. The recoveries of tap water and lake water were observed in the range of 93.1–105.6%, which demonstrated low matrix effect on the amperometric response. The low relative standard deviations for chlorpyrifos and carbaryl demonstrated the high precision of analysis

Conclusion

In this work, combining the advantageous characteristics of Ag NPs and CGR, NF and CS, a novel AChE biosensor based on Ag NPs–CGR–NF has been developed. 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 carbaryl 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.

Reference:                                                       

Liu, Y., Wang, G., Li, C., Zhou, Q., Wang, M. and Yang, L., 2014. A novel acetylcholinesterase biosensor based on carboxylic graphene coated with silver nanoparticles for pesticide detection. Materials Science and Engineering: C, 35, pp.253-258.