Preparation of Ag-Fe3O4 nanoparticles sensor and application in detection of methomyl

Ag-doped Fe3O4 nanoparticles (Ag-Fe3O4) was successfully synthesized by the hydrothermal method and performed a simple chemical reaction process in a significantly shorter time than traditional solvothermal method. Furthermore, Ag-Fe3O4 electrochemical sensor was prepared using chitosan and acetic acid as crosslinkers, and the electrochemical behavior of methomyl on the electrode was studied by cyclic voltammetry. Experimental results showed that the electrode had faster response, higher detection sensitivity and better stability for methomyl. Under optimal conditions, the linear current response was achieved in the concentration range of 2.97×10-5 mol·L-1～3.47×10-4 mol·L-1, with the detection limit of 2.08×10-5 mol·L-1. The methomyl in different vegetable samples was detected, and its recovery rate was between 90%～98%.


Introduction
Carbamate pesticides are synthetic pesticides developed after organophosphorus pesticides. Because of its characteristics of strong selectivity, high efficiency, broad spectrum, low toxicity to humans and livestock, easy decomposition and low residual toxicity, it has been widely used in agriculture, forestry, and animal husbandry. However, large amounts of residual carbamate pesticides remain in the environment, and lead to serious water pollution. Residual pesticides entered the human bodies along with the food chain. As a result, cholinesterase activity could be inhibited and the hydroxyl group of serine in the enzyme active center could be carbamylated. More seriously, enzymes would lose the hydrolysis ability of acetylcholine and cause poisoning due to the accumulation of acetylcholine in the tissues. Even cholinergic characteristic of tears, salivation, pupils dilate, convulsions and death would occur. Therefore, residues of carbamate pesticides are detected timely and accurately ， which is of great importance in developing and using pesticides rationally, and protecting the ecological environment and human health.
Traditional detecting methods of residual pesticides include high performance liquid chromatography [1], gas chromatography/mass spectroscopy [2], spectrophotometry [3], chemiluminescence [4] and enzymatic inhibition method [5]. Although these methods can accurately detect content of residual pesticides, the equipment is expensive and the sample pretreatment methods are tedious with long analysis period. Recently, with the rapid development of electrochemical sensing technology, more researchers are committed to developing a rapid, sensitive and simple carbamate pesticide sensor used in on-site monitoring.
Magnetic Fe 3 O 4 nanoparticles have good biocompatibility. Their special electronic configuration and chemical bonding activity are applied to biosensors such as immune sensors and DNA sensors to promote the electron transport. Recently, a lot of work has been done to modify the surface of Fe 3 O 4 nanoparticles to produce different kinds of composite nanoparticles in many experiments. The present paper shows the nanosilverdoped magnetic Fe 3 O 4 nanoparticles could improve the stability of nanoparticles and the synthesized Ag-Fe 3 O 4 nanoparticles was applied to the preparation of electrochemical sensors. Therefore, a method for detecting carbamate residual pesticides was established.
Methomyl is an N-methyl carbamate saccharide insecticide of all carbamate pesticides. In this paper, Ag-Fe 3 O 4 /GCE (glassy carbon electrode) chemical sensor was constructed by the characteristics of Ag-Fe 3 O 4 composites, and its electrochemical behavior of methomyl on the electrode was measured for further measuring the carbamate pesticide residues in agricultural products. P) were all used in this study, and these chemicals were commercially avaliable and of analytical grade. The ultra-pure water was used. Spinach, oilseed rape and lettuce was purchased from the backdoor market in Longdong University.

Preparation of nano Ag
In this typical experiment, 0.1 g soluble starch, 2~3 drops of 0.05 mol•L -1 silver nitrate aqueous solution, 0.02 g dihydrate of trisodium citrate and 0.03 g glucose was successively put into 25 mL ultra-pure water in a small beaker and mixed evenly [6]. After the reaction, liquid was taken to test in a transparent tube and heated for 3 minutes until boiling in a household microwave oven. Finally, the buff nano Ag solution was successfully prepared.

Preparation of nano Fe3O4
At room temperature, 120 mL of 1.5 mol•L -1 sodium hydroxide aqueous solution was put into a four-necked flask and then heated to 80 °C. Fe 2 (SO 4 ) 3 •XH 2 O (4.3 g), Fe 2 SO 4 •7H 2 O (1.5 g) and H 2 SO 4 (2 mL, 2 mol•L -1 ) was mixed with 20 mL ultra-pure water thoroughly, and then the mixture was gradually dripped into NaOH solution at 80 °C in the four neck flask to react for 1 h. Finally, magnetic Fe 3 O 4 nanoparticles were obtained through magnetic separation [7].

Preparation of Ag-Fe3O4 composites
Trisodium citrate aqueous solution (145 mL, 11.0×10 mol•L -1 ), sodium dodecyl benzene sulfonate aqueous solution (1 mL, 0.7 mol•L -1 ) and the as-prepared magnetic Fe3O4 nanoparticles (0.15 g) was put into a four-necked flask. While the mixed solution was heated to keep in a slightly boiling state, 50 mL of 4×10 -2 mol•L -1 silver nitrate aqueous solution was quickly dropwise added into the flask. Then the final mixture in the flask was mechanically stirred for 30 minutes. Finally, magnetic Fe3O4-Ag nanocomposite product was obtained by magnetic separation and dried in a vacuum [7].

Preparation of electrochemical sensors
The bare gold electrode was polished to mirror on chammy with 0.3 μm and 0.05 μm Al2O3 pasted and then treated ultrasonically in the secondary water to obtain a smooth electrode surface. Before the modification, the gold electrode was scanned in the mixture of potassium ferricyanide and potassium nitrate until a stable cyclic voltammogram was obtained. At the end of the experiment, the electrode must be ground clean, rinsed with ultra-pure water and ethanol, dried and placed for next use [8][9].
A certain amount of dispersion solution of Ag-Fe3O4 nanoparticles was ultrasonicated for 2 minutes. The above solution (5 μL) taken by the feeder was dripped onto the surface of the dried and pretreated bare gold electrode and dried naturally (or placed in a 37 °C incubator) [10]. Then 0.02 g of chitosan (CHIT) was mixed with 1 mL of acetic acid solution evenly. Such mixture (5 μL) was dripped onto the gold electrode modified with a solution of Ag-Fe3O4 nanoparticles and got dried in the air. Finally, an Ag-Fe3O4/GCE chemical sensor was obtained.

Characterization
The morphology of the as prepared nanocomposite was identified with a JEOL JSM-6510LV and JEOL6700-F of scanning electron microscope (SEM) and transmission electron microscopy (TEM, FEI Tecnai G20). The ultraviolet-visible (UV-vis) diffuse reflectance spectra of the samples were obtained for the dry-pressed film samples by a UV-vis spectrophotometer (SPECORD50 PLUS, Analytik Jena). BaSO 4 was used as a reflectance standard in a UV-vis diffuse reflectance experiment. In addition, the test instruments include electronic balance (BS110S), PHS-3C ray magnetic pH meter, PJ25B-A microwave oven, KQ3200 ultrasonic processing equipment, quartz UV cuvette, RST electrochemical workstation (Zhengzhou Cirusi instrument technology co., LTD.) and DHG-9140A electric thermostatic blast drying oven (Shanghai Yiheng technology co., LTD.).

Results and Discussion
The optical properties of the Ag-Fe3O4 composites were explored by UV-vis diffuse reflectance spectra (DRS).  The absorption edge of the Ag-Fe3O4 was shifted to a longer wavelength compared with that of pure Ag due to the contribution of black Fe3O4. Therefore, the observation in the UV-Vis diffuse reflectance spectra indicates that Ag nanoparticles were successfully coated on the surface of Fe3O4 particles.
The microscopic morphologies of Ag-Fe 3 O 4 composites and pure Fe 3 O 4 nanoparticles were determined by scanning electron microscopy (SEM) and transmitting electron microscopy (TEM). The pure Fe 3 O 4 nanoparticles were investigated. As shown in Fig.2(b,c), Fe 3 O 4 is distinct from lattice fringes. However, because of the magnetic effect, Fe 3 O 4 nanoparticles were easily agglomerated and difficult to disperse evenly in water. Fig 2(a,b) shows a typical SEM image of Ag-Fe 3 O 4 composites which was composed of irregular small blocks, and the surface was attached with many small particles. The TEM image further confirms this phenomenon. As shown in Fig 2(d,e), the Ag-Fe 3 O 4 composite nanoparticles resemble the core-shell, and the surface of Fe 3 O 4 was coated with a layer of silver particles and dispersed more evenly. It can be found that the Ag nanoparticles effectively inhibited the agglomeration of the magnetic Fe 3 O 4 nanoparticles and improved the stability of these nanoparticles. With the Ag-Fe 3 O 4 /GCE as working electrode, the saturated calomel electrode (SCE) as reference electrode, platinum wire electrode as counter electrode, different concentrations of methomyl with phosphate buffer solution (0.1 mol•L -1 ，pH=6.9) were scanned several times through cyclic voltammetry for detecting the electrochemical properties of different concentrations of methomyl. Until the curve was stable, the cyclic voltammogram was obtained to record peak current and corresponding potential. Fig.3 is the cyclic voltammogram obtained by a scanning concentrations of 2.97×10 -5 mol•L -1 ～3.47×10 -4 mol•L -1 methomyl. It has been found that when the concentration of methomyl was 1.04×10 -4 mol•L -1 , its electrochemical performance is optimal.

Conditional optimization experiment
The effect of pH value of phosphate buffer solution on the peak current response of Ag-Fe 3 O 4 /GCE electrode was studied in this experiment. When the pH value of phosphate buffer solution was 6.0 to 7.0, the peak current of Ag-Fe 3 O 4 /GCE chemical sensor in the concentration of 1.04×10 -4 mol·L -1 methomyl prepared with phosphate buffer solution was measured to find the optimal pH value of phosphate buffer solution. The peak current value of the buffer solution was optimum at a pH value of 6.85 to 7.0 (Fig.4). Carbamate pesticides were relatively stable in acidic environment and easily decomposed when exposed to alkalis, so the pH value of 6.9 was chosen as the acidity of this experiment [11]. During the modification of the Ag-Fe 3 O 4 /GCE electrode, the effect of the Ag-Fe 3 O 4 nanoparticle solution dosage on the peak current response of this electrode was discussed in this experiment. When the dosage of solution was 3～15 μL, the peak current of Ag-Fe 3 O 4 /GCE chemical sensor in a series of values selected were measured respectively at a pH value of 6.9 and a concentration of 1.04×10 -4 mol·L -1 methomyl, and the optimum amount of modification was determined. When less Ag-Fe 3 O 4 nanoparticles were immobilized on the electrode, the response current was small, and maybe too small to completely react with methomyl, so the response was not sensitive enough (Fig.5). With the increment of coating drops, the peak current value became larger and reached the maximum at 5 μL. When the amount of coating drops continued to increase, the response current tended to decrease because the drop coating was too thick, which caused greater resistance. After reaching 8 μL, it started to increase again, and the effect of the resistance was negligible. After reaching 8 μL coating drops, the peak current value increased, but the surface of the electrode was very limited. When the amount of coating was too large in the dispensing, there was partial loss and the measurement was inaccurate. Therefore, 5 μL of the modification coating drops was selected in this experiment appropriately. The scan rate had a great influence on the peak current. A good linear relationship was presented between the reduction peak current and the square root of scan rate within a certain range (Fig.6). Therefore, the linear regression equation was I=13.48-70.72V 1/2 , and the linear coefficient R was 0.9967. It shows that the mass transfer process of the electrode was controlled by diffusion. When the scan rate was too high, the background current became larger and could interfere the measurement of peak current. In this experiment, 0.1 V/s was taken as the optimal scan rate with the best signal-to-noise ratio [12][13]. Under optimized experimental conditions, the methomyl solution diluted 100 times was further diluted 35 times, 50 times, 100 times, 150 times, 200 times, 250 times, 300 times, and 350 times for tests. (The concentration of methomyl stock solution was 1.04 mol•L -1 ). Different peak currents at different concentrations of methomyl were measured experimentally to obtain a working curve for methomyl pesticides (Fig.7). I = 0.3973C+ 0.412 (C was 1.04×10 -4 mol•L -1 ) , R 2 = 0.9903.

Sample determination
The edible part of lettuce, oilseed rape and spinach was selected and cut into pieces with a knife, and then accurately weighed 10 g into two erlenmeyer flasks. Phosphate buffer solution (50 mL) was added in one erlenmeyer flask, and acetone (5 mL) and phosphate buffer solution (45 mL) was added in other erlenmeyer flask. After soaking shreds for about 12 h, the soaked liquid was followed by the sonucation for 5 minutes. Then the liquid was stood for a while. Finally, the supernatant was poured out for further use.
The liquid phase of lettuce (1.0 mL), oilseed rape (2.5 mL), and spinach (5 mL) respectively was transfered into a volumetric flask in turn. Under the optimized experimental conditions, added phosphate buffer to scale respectively. Finally, added the standard solution of methomyl, measured 3 parallel tests, took the average value and calculated the recovery rate.
The peak currents of the above samples (1 mL of lettuce test solution, 2.5 mL of oilseed rape test solution, and 5 mL of spinach test solution) were measured in parallel for 3 times by cyclic voltammetry to compare with the above standard curve, and then the residual amount of their pesticide was obtained. It was calculated that the pesticide concentration in spinach, oilseed rape and lettuce was respectively 0.1099×10 -4 mol•L -1 , 0.4996×10 -4 mol•L -1 and 0.4276×10 -4 mol•L -1 . The standard addition method was used to estimate the accuracy of this method. After adding 1.04×10 -4 mol•L -1 methomyl standard solution into the test solution, its peak current value was 4.9131×10 -6 A. Then the average value of 3 parallel tests was taken and the recovery rate was calculated. {recovery rate=[(standard sample test value -sample test value) / (the adding standard amount)]×100%}. The results are shown in Table 1. From the recovery rates in Table 1, it could be seen that methomyl was completely recovered by this method, so it was feasible for detection the methomyl residuals.

Measurement of interference experiment
This experiment investigated the interference of three organophosphorus pesticides on methomyl. 1 mL stock solution of each of the three pesticides of dichlorvos, chlorpyrifos and phoxim was diluted 5,000 times, and then added into 1.04×10 -4 mol•L -1 of methomyl standard solution. The peak current value of the above solution was measured through cyclic voltammetry to compare with the standard solution [14]. Experiment results show that dichlorvos, chlorpyrifos and phoxim would interfere with the determination of methomyl.

Conclusions
In this method, a new electrochemical sensor Ag-Fe 3 O 4 /GCE was prepared, and the electrode response of this sensor to different concentrations of methomyl solution was determined in a phosphate buffer solution with a pH value of 6.9. The pesticide residues of methomyl in vegetables determined by this sensor was used to establish a method for the determination of carbamate pesticides in agricultural products. It is simple, fast, responsive, low sample dosage and non-destructive to the samples, and it may have broad development prospects in the future.