Graphene oxide / multi-walled carbon nanotubes / gold nanoparticle hybridfunctionalized disposable screen-printed carbon electrode to determine Cd ( II ) and Pb ( II ) in soil

Cadmium (Cd) and lead (Pb) in soil or water environment cause the ecological destruction and environmental deterioration when their contents exceed the natural background values. To trace the concentrations of Cd(II) and Pb(II), a sensitive and selective electrode was developed using disposable screen-printed carbon electrode (SPE) immobilized with a composite film of reduced graphene oxide/carboxylation multi-walled carbon nanotubes/gold nanoparticle hybrid (RGO-MWNT-AuNP) through π-π bind. This highly conductive nano-composite layer, “RGO-MWNT-AuNP,” was characterized by scanning electron microscopy, UV-visible spectrometer, cyclic voltammetry, and electrochemical impedance spectroscopy. Square wave stripping voltammetry was applied to RGO-MWNT-AuNP/SPE to electroplate bismuth film and monitor the Cd(II) and Pb(II) simultaneously. To obtain high current responses, the detecting parameters were optimized. Under optimized conditions, the current responses showed a linear relationship with the concentrations of Cd(II) and Pb(II) in the range from 1.0 to 80.0 μg/L with a lower detection limit of 0.7 μg/L and 0.3 μg/L (S/N = 3), respectively. Finally, the prepared electrode was further employed to detect Cd(II) and Pb(II) in soil samples with good results.


Introduction 
Heavy metals, including mercury (Hg), cadmium (Cd), lead (Pb), chromium (Cr), Copper (Cu) and Arsenic (As) [1] , are widely distributed in natural environment, which have serious hazards to human health and proved by many researches.Lead ion (Pb(II)) and cadmium ion (Cd(II)), which are two of the most common contaminants, attracted stronger interest in China due to their vast emissions and persistent environmental pollution [2,3] .In the agricultural environment, Pb(II) and Cd(II) in soil or water are mainly derived from wastewater discharge, vehicle emissions, lead-acid batteries, fertilizers, mining and industrial wastes [1,4] .According to a nationwide survey conducted by the Chinese government which evaluated 6.3 million km 2 of various land from 2005 to 2013, half of these land was suffering contamination from heavy metals especially lead and cadmium in varying degrees, caused a severe threat to food safety, ecological environment, and sustainable agricultural development.These two heavy metals in farmland soil are easily absorbed by crops and vegetables, which can be entered and enriched in human body through multiple absorption ingestion and dermal contact.Pb(II) and Cd(II) that binded with enzymes, nucleic acids and structural proteins will interfere, impact and destruct the body's normal function [5] , and they can further cause server diseases such as pneumonitis, lung cancer, osteomalacia, anemia, encephalopathy [6] .To assess the levels of Pb(II) and Cd(II) in soil, a cheap and convenient analytical tool is urgent to develop for heavy metal ions determination.Currently, many methods have been exploited and applied in testing organizations during the past decades, including atomic absorption spectrometry [7] , inductively coupled plasma mass spectrometry [8,9] , capillary electrophoresis [10] , X-ray fluorescence spectrometry [11] , and quantum dot [12] .Most of these techniques have high sensitivity and selectivity, but they required bulk and expensive instrument, sophisticated operation and complex pretreatment that make them difficult to detect heavy metal ions on-site.Electrochemical method [13] has gained a widespread attention in recent years because of its simple operation, small device and inexpensive electrodes.Carbon paste electrode and glassy carbon electrode are the common working electrodes in electrochemical sensing, but their price is relatively higher than disposable screen-printed electrode (SPE) [14] .
Besides, SPE integrated three electrodes (a working electrode, a reference electrode, and a counter electrode) on a substrate, owns some significant advantages that can be produced in enormous quantities.However, bare SPE has many drawbacks including low sensitivity and poor selectivity, which makes it hard to trace the super-low level of Cd(II) and Pb(II).
Several new materials, such as nano materials [14] , conductive polymer [15] , ionic liquids [16] and biologic material [17] , have been applied in the field of electrochemistry to improve the detecting performance.Biologic materials containing proteins, nucleic acids and cells can bind Cd(II) and Pb(II) with high affinity and specificity, but they are more susceptible to the environment which limits their application.Nanomaterials like graphene [18] and carbon nanotubes [19] have many unusual characters such as physicochemical, mechanical, excellent electrical, optical and thermal conductivity, which makes them been applied in many fields.Some researchers tried to combine graphene and carbon nanotubes.They found that carbon nanotubes can connect with graphene to bridge the defects for electron transfer, and these hybrid materials have a unique synergistic effect of graphene and carbon nanotubes [20,21] .Graphene oxide [22] is an oxygenated derivative of graphene which has excellent hydrophobicity, which makes it easily form a homogeneous liquid with water to provide active sites for the interaction with other aromatic compounds.Gold nanoparticles (AuNP) have many special physical and chemical properties including large specific surface area, high surface energy, quantum dimension effect and macroscopic quantum tunneling effect.
Screen-printed carbon electrode (SPE) is a low cost, small-size, portable and easy-to-use electrode, which have been modified with different chemical materials and biomaterials to improve the specificity and sensitivity.
This study synthesized a nano-composite material using graphene oxide, multi-wall carbon nanotube and gold nano particles, which was used to modify a disposable SPE to overcome the poor detecting performance.After GO reduced by cyclic voltammetry, RGO-MWNT-AuNP/SPE is plated a bismuth film in situ that obtains the current signal of Cd(II) and Pb(II) at the same time.The properties of SPE before and after each modification were investigated by some methods.After optimized the electroanalytical parameters, the modified SPE was employed to detect the Cd(II) and Pb(II) using square wave stripping voltammetry.

Chemicals and reagents
Three standard solutions of Bi(III), Cd(II) and Pd(II) (1000 mg/L) were offered by National Standard Reference Materials Center (Beijing, China), which were diluted as required before use.Graphene oxide and multi-walled carbon nanotube were bought from Xianfeng Nano-Materials Technology Co., Ltd.(Nanjing, China).HAuCl 4 were purchased from Aldrich (Sigma-Aldrich US).
Potassium ferricyanide was obtained from Shanghai Chemical Reagent Co. (Shanghai, China).Sodium phosphate buffer solution was employed as supporting electrolyte to reduce GO-MWNT-GNP hybrid.Sodium acetate buffer solution was took as the supporting electrolyte for the Cd(II) and Pb(II) determination.The rest of chemicals that not mentioned here were of analytical reagent grade and were used as received.All solutions were prepared using Milli-Q purified water (>18.2MΩ/cm 3 ).

Instrument
All electrochemical measurements were conducted using CHI 660D electrochemical workstation, which were purchased from the CHI Instrument Company (Shanghai, China).Three electrodes were integrated on the same substrate (Bi/RGO-MWNT-AuNP/SPE as the working electrode, printed Ag electrode as the reference electrode and printed carbon electrode as the auxiliary electrode).A PHS-3C digital pH-meter was purchased from Shanghai Instrument Scientific Instrument Co., Ltd, China.UV-vis experiments were measured using a UV-2100S spectrophotometer (Shimadzu).The images of scanning electron microscopy (SEM) were achieved by a field-emission scanning electron microscope JEOLJEM-2011 device.All measurements were carried out at room temperature.

Synthesis of GO-MWNT-GNP and AuNP
The GO-MWNT hybrid was synthesized using the protocol reported in previous report [23] .Briefly, MWNT were refluxed with mixture of concentrated HNO 3 and concentrated H 2 SO 4 (V/V=1:3) for 6 h.The carboxylation of MWNT(c-MWNT) was filtered and rinsed with sufficient Milli-Q purified until neutral pH was achieved.Additionally, the c-MWNT were dried at high temperature in a vacuum oven. 1 mg c-MWNTs were dispersed in 1 mL Milli-Q purified water to form a homogeneous solution, and then the dispersed solution was mixed with 1 mL GO (1 mg/mL) suspension, which was further sonicated for 0.5 h to form a uniform GO-MWNT complexes.
The AuNP were synthesized using citrated reduction method [24] .Before the experiment, all glasswares were immersed in aqua regia and rinsed with sufficient water to remove the potential artificial nucleation sites.50 mL of 1 mmol/L tetrachloroaurate trihydrate solution was boiled with vigorous stirring until the temperature reached 97°C, and 1.5% trisodium citrate was then added to the boiling solution as soon as possible.The mixed solution stops stirring when its color turns brilliant red.

Electrode preparation
The GO-MWNT-AuNP hybrid was prepared by mixing 20μL GO-MWNT, 20 μL AuNP, 50 μL water and 10 μL ethyl alcohol.4 μL GO-MWNT-AuNP hybrid was coated on the working electrode surface, which was dried in air for 30 min to obtain GO-MWNT-AuNP/SPE.After that, GO-MWNT-AuNP/SPE was immersed in 0.1 mol/L PBS (pH 7.0) and reduced the GO to gain RGO-MWNT-AuNP/SPE through cyclic voltammetry with scan rate 20 mV/s as shown in Figure 1.

Measurement procedures
For the Cd(II) and Pb(II) determination, the measurements using the square wave anodic stripping voltammetry (SWASV) were performed in 0.1 mol/L sodium phosphate buffer solution with the presence of 1 mg/L Bi(III).Before the analysis, three electrodes were immersed in the extract of soil samples, and a negative potential (-1.4 V) was applied to the working electrode with stirring in the test solution during the deposition steps.After a 20 s equilibration period, the potential scan of SWASV was scanned from -1.4 V to +0.0 V.The microstructure of the working electrode surface was investigated via the scanning electron microscope (SEM).Figure 2 shows the SEM images of SPE, RGO-MWNT/SPE, and RGO-MWNT-AuNP/SPE.The morphology of SPE in Figure 2(a) exhibited some large irregular stones gaps and graphite blocks.After SPE functionalized with RGO-MWNT hybrid, RGO and MWNT were homogeneously distributed on the working electrode surface, which owns a large specific surface area due to the special structure of RGO and MWNT.From the SEM image of RGO-MWNT-AuNP/SPE, there were too many tiny gaps distributed on the working surface that can provide large interface compared to RGO-MWNT, which was contributed to AuNP.
UV-vis spectroscopy was employed to analyze the dispersed solution of different materials.From the Figure 3, it was obvious that the UV-vis spectrum of AuNP in water displayed two absorption bands at 250 nm and 540 nm, and the absorption band of GO-MWNT was located at 240 nm.When these two materials were mixed, the UV-vis spectrum of this new hybrid shows a small absorption band at 540 nm and a high absorption band at 250 nm.The results seem like the sum of the absorption of AuNP and GO-MWNT, indicating that these two material form a homogeneous solution.EIS was a useful method to characterize the impedance changes of SPE before and after modification, which was consisted by a semicircle part at higher frequencies and a linear part at the lower frequencies that presented the electron transfer resistance (R et ) and the diffusion process, respectively.In Figure 5, the R et value of SPE was about 2100 Ω.After RGO-MWNT was immobilized on SPE, the conductivity between RGO-MWNT/SPE and electrolyte was enhanced, and the R et value decreased to 1500 Ω.Similarly, R et value of the RGO-MWNT-AuNP/SPE was about 1100 Ω due to the synergistic effect of AuNP and RGO-MWNT.The results were in agreement with the conclusion obtained from the CV.The potential window of RGO-MWNT-AuNP/SPE before and after electroplating Bi film was tested in Figure 6.
The RGO-MWNT-AuNP/SPE without bismuth film had a narrow potential window, which was greater -1.2 V.If the potential was lower than -1.2 V, the current response was increased significantly because of the hydrogen evolution occurring on the electrode surface.After reducing a Bi film, Bi/RGO-MWNT-AuNP/SPE can obtain a relative negative hydrogen over voltage potential about -1.6 V, which was ascribed to the special crystal plane structure of bismuth.Bi film decorated one lectrodes in-site is used to improve the sensitivity for the detection of Cd(II) and Pb(II) because Bi(III) can form a complex with Cd(II) and Pb(II) that makes their concentration reduced efficiently.Figure 8a displays the current response of Cd(II) and Pb(II) associated with the Bi(III) concentration.It was obvious that the current responses of Cd(II) and Pb(II) were of positive relevance with Bi(III) concentrations ranging from 0 to 1 mg/L.The adequate Bi(III) with Cd(II) and Pb(II) can form a complex to decrease the activation energy for cathodic preconcentration.When the Bi(III) concentration was higher than 1 mg/L, the current responses of Cd(II) and Pb(II) decreased.
Thus, 1 mg/L of Bi(III) was chosen in the experiments.
Figure 8b shows that the current responses of Cd(II) and Pb(II) were affected by the pH of the sodium acetate buffer solution.Different pH in the range from 3.5 to 5.5 was investigated for the Cd(II) and Pb(II) detection.The highest current responses of Cd(II) and Pb(II) were located at pH4.5.The reason was that hydrogen generated at excessively acidic circumstances on electrode' surface can damage the quality of the bismuth film.In contrast, the current responses remained low due to the hydrolysis of Bi(III) at higher pH.Hence, pH4.5 was used throughout the experiments.
Figure 8c shows the deposition potential associated with the current response of Cd(II) and Pb(II).The current responses of Cd(II) and Pb(II) were enhanced with the deposition potential ranging from -1.2 to -1.4 V, and then decreased remarkedly when the deposition potential was higher than -1.4 V, which was attributed to the hydrogen evolution reaction that prevented the deposition of Cd(II) and Pb(II) on the surface.Therefore, -1.4 V was chosen as the optimized deposition potential.
Figure 8d shows that the current response was affected by the deposition time.The peak currents increased with the deposition time from 60 to 420 s.However, the peak currents increased gradually when the deposition time exceeded 300 s.With the consideration of the rapid detection, 300 s was selected as the optimized deposition time.Compared with heavy metal electrodes reported by other groups, some properties for Cd(II) and Pb(II) detection were summarized in Table 1.It was obvious that the linear range of Bi/RGO-MWNT-AuNP/SPE can reach the performance of the modified glassy carbon electrode, but the low detection limit was much higher than others.The proposed electrode can meet the requirements of the Cd(II) and Pb(II) detection in soil.

Interference effects
The performance of anti-interference was an important parameter for the electrochemical electrode.
First, the Bi/RGO-MWNT-AuNP/SPE was employed to test the standard solutions of Cd(II) and Pb(II) in Table 2, indicating that Bi/RGO-MWNT-AuNP/SPE had high accuracy.Second, the modified electrode was also used to test 10 µg/L Cd(II) and 10 µg/L Pb(II) with 10-fold co-existing ions (K(I), Cl(I), Na(I), NO 3 (I), CH 3 COO(I), Mg(II), Ca(II), Co(II), Ni(II), SO 4 (II), Fe(III) and Al(III)) under the optimal conditions.The results show that the relative currents of most metal ions were lower than 5%, illustrated that these cations and anions did not interfere with Cd(II) and Pb(II) determination.

Analysis of the soil samples
To estimate its application, Bi/RGO-MWNT-AuNP/SPE was employed to sense Cd(II) and Pb(II) in extracting soil solutions.All soil samples were collected from farmland in the suburbs of Beijing city.
Before measurement, the soil samples were pretreated to extract Cd(II) and Pb(II) [31] .First, soil samples were heating to dry in an oven at a high temperature.Then, the dried samples were ground using a mortar. 1 g of soil was transferred to an extraction bottle and mixed with 0.1 mol/L acetic acid, and the mixture was processed using ultrasonic treatment for 2 h at normal temperature.After a period of static conditions, the supernatant liquid was centrifuged, and the supernatant was filtered through a 0.22-μm membrane.Finally, the pH was adjusted to 4.5 by the addition of sodium acetate buffer solution.
Bi/RGO-MWNT-AuNP/SPE and flame atomic absorption spectrometry (FAAS) were employed separately to measure the Cd(II) and Pb(II) in the extract solutions of soil.Then, the extract solutions were spiked with different Cd(II) and Pb(II) concentrations and measured by Bi/RGO-MWNT-AuNP/SPE again.The results were shown in Table 3.It can be seen that the recovery of Cd(II) and Pb(II) were ranging from 91.98% to 105.36%, which can meet the requirement of rapid detection on-site.

Conclusions
In summary, a nano-composite material was synthesized by GO, MWNT, and AuNP, which exhibits unique structural features.The electrochemical characteristics were improved significantly when bare SPE was modified with this new composite material.The RGO-MWNT-AuNP/SPE electroplated the Bi film ex-site can detect Cd(II) and Pb(II) simultaneously with high sensitivity.This prepared electrode has shown excellent anti-interference and can be employed to measure field sample with high accuracy.

Figure 6 Figure 7
Figure 6 Cyclic voltammograms of RGO-MWNT-AuNP/SPE and Bi/RGO-MWNT-AuNP/SPE in 0.1 mol/L acetate buffer solution Figure 7 shows the SWASV responses of Bi/SPE, Bi/RGO-MWNT/SPE, and Bi/RGO-MWNT-AuNP/SPE, which were used to detect 20 µg/L Cd(II) and 20 µg/L Pb(II) in 0.1 mol/L acetate buffer solution.Two reductive peak currents for the Cd(II) and Pb(II) determination was observed at -1.1 V and -0.85 V on the SPE.The change of reductive potentials was mainly because the reference electrode of Ag/AgCl was replaced by the Ag electrode, and the electrode potentials of these two reference electrode were different.Bi/RGO-MWNT/SPE and Bi/RGO-MWNT-AuNP/ SPE exhibited much higher current responses toward Cd(II) and Pb(II) determination in comparison to SPE's.The reason was that nano-composite film of RGO-MWNT and RGO-MWNT-AuNP

Figure 7
Figure 7 SWASV responses of (a) Bi/SPE, (b) Bi/RGO-MWNT/SPE and (c) Bi/RGO-MWNT-AuNP/SPE in 0.1 mol/L acetate buffer solution with 20 μg/L Cd(II) and 20 μg/L Pb(II) 3.3 Optimization To improve the current response for Pb(II) and Cd(II), several criticaldetecting parameters of Bi/RGO-MWNT-AuNP/SPE were optimized in 0.1 mol/L sodium acetate buffer solution, which contained 50 μg/L Cd(II) and 50 μg/L Pb(II).Bi film decorated one lectrodes in-site is used to improve the sensitivity for the detection of Cd(II) and Pb(II) because Bi(III) can form a complex with Cd(II) and Pb(II) that makes their concentration reduced efficiently.Figure8adisplays the current response of Cd(II) and Pb(II) associated with the Bi(III) concentration.It was obvious that the current responses of Cd(II) and Pb(II) were of positive relevance with Bi(III) concentrations ranging from 0 to 1 mg/L.The adequate Bi(III) with Cd(II) and Pb(II) can form a complex to decrease the activation energy for cathodic preconcentration.When the Bi(III) concentration was higher than 1 mg/L, the current responses of Cd(II) and Pb(II) decreased.Thus, 1 mg/L of Bi(III) was chosen in the experiments.Figure8bshows that the current responses of Cd(II) and Pb(II) were affected by the pH of the sodium acetate buffer solution.Different pH in the range from 3.5 to 5.5 was investigated for the Cd(II) and Pb(II) detection.The highest current responses of Cd(II) and Pb(II) were located at pH4.5.The reason was that hydrogen generated at excessively acidic circumstances on electrode' surface can damage the quality of the bismuth film.In contrast, the current responses remained low due to the hydrolysis of Bi(III) at higher pH.Hence, pH4.5 was used throughout the experiments.Figure8cshows the deposition potential associated with the current response of Cd(II) and Pb(II).The current responses of Cd(II) and Pb(II) were enhanced with the deposition potential ranging from -1.2 to -1.4 V, and then decreased remarkedly when the deposition potential was higher than -1.4 V, which was attributed to the hydrogen evolution reaction that prevented the deposition of Cd(II) and Pb(II) on the surface.Therefore, -1.4 V was chosen as the optimized deposition potential.Figure8dshows that the current response was affected by the deposition time.The peak currents increased with the deposition time from 60 to 420 s.However, the peak currents increased gradually when the deposition time exceeded 300 s.With the consideration of the rapid detection, 300 s was selected as the optimized deposition time.

Figure 8
Figure 8 SWASV responses for 50 μg/L Cd(II) and 50 μg/L Pb(II) detection in 0.1 mol/L acetate buffer solution affected by (a) bismuth concentration, (b) pH, (c) deposition potential and (d) deposition time 3.4 Analytical performance of Bi/RGO-MWNT-AuNP/SPE The calibration curves for Cd(II) and Pb(II) determination were performed on the Bi/RGO-MWNT-AuNP/SPE under the optimal conditions.The electrode was immersed in 0.1 mol/L acetate buffer solution (pH 4.5) by adding the standard solution.Figure 9 shows a series of current responses for different concentrations of Cd(II) and Pb(II).It was found that the current responses and the concentrations of Cd(II) and Pb(II) exhibited a linear relation in the range from 1 μg/L to 80 μg/L.The regression equations were: I(μA)=0.4413×C(μg/L)+0.3001and I(μA) =0.4893× C(μg/L)+0.6285with the correlation coefficients

Figure 9
Figure 9 SWASV curves of the Bi/RGO-MWNT-AuNP/SPE to detect different Cd(II) and Pb(II) concentrations ranging from 1 μg/L to 80 μg/L in 0.1 M sodium acetate buffer solution; (A) The calibration curve of Cd(II); (B) The calibration curve of Pb(II)