Stripping Voltammetric Determination of Lead in Coastal Waters With a Functional Micro-Needle Electrode

In this work, for the voltammetric determination of lead (Pb) in coastal waters, a novel functional micro-needle electrode based on 3D reduced graphene oxide/flower-like bismuth nanosheets (3DrGO/F-BiNSs) was fabricated. The micro-needle electrode was prepared with the commercial acupuncture needle (ANE) and functionalized with 3DrGO/F-BiNSs through stepwise electrodeposition method. The sensing surface (needle tip) of the micro-needle electrode was first modified with 3DrGO to enhance the conductivity and provide more active sites to combine Bi nanomaterials. Then the F-BiNSs were electrodeposited on the 3DrGO surface to facilitate the electrochemical voltammetric response of Pb2+ due to their excellent combining ability with Pb. The physical and electrochemical properties of the as-prepared 3DrGO/F-BiNSs/ANE were characterized by different techniques including scanning electron microscope (SEM), energy dispersive X-ray spectroscopy (EDS), and electrochemical methods. The 3DrGO/F-BiNSs/ANE exhibited an excellent performance for the voltammetric determination of Pb2+ with the linear range of 40 to 600 nmol L–1 and detection limit of 12.5 nmol L–1. Additionally, the application of the functional micro-needle electrode for Pb determination in different coastal water samples was also investigated.


INTRODUCTION
In the past decades, with the development of global economy and industry, much attention has been attracted to the marine environmental pollution problems. Lead (Pb), one of the heavy metal ions, is highly toxic to the marine aquatic plants and animals and eventually harmful to the human beings through bioaccumulation (Kazi et al., 2009;Chang et al., 2014). It has been reported that the content of Pb in coastal waters is much higher than that in the open ocean because of the enormous amount of anthropogenic contamination (Zhang et al., 2016). Therefore, determination of Pb 2+ in coastal waters is of great importance for the environmental protection and public health. Many techniques have been developed for the determination of heavy metal ions, including atomic absorption spectroscopy, atomic emission spectroscopy, and inductively coupled plasma-mass spectrometry (Manivannan and Biju, 2011;Zhu et al., 2017). Although these methods possess various advantages and have been commonly used, the requirement of cumbersome instruments, time-consuming pretreatments, and high cost are the inevitable limitations (Hu et al., 2015;Zhang et al., 2016).
As an efficient electrochemical technique for the determination of heavy metals, anodic stripping voltammetry (ASV) has attracted much attention due to its advantages, such as high sensitivity, rapid response, and straightforward analytical processes (Aragay et al., 2011b;Wang et al., 2014;Lin et al., 2016). In the past decades, mercury electrode, such as hanging mercury electrode, dropping mercury electrode, and mercury film electrode has been commonly adopted for the ASV determination of heavy metals (Nedeltcheva et al., 2005;Annibaldi et al., 2015;Granado-Castro et al., 2018). However, the drawbacks of serious toxicity and difficulties in handling process limited the continued widespread application of mercury electrode (Nagai et al., 2004;Guzzi and La Porta, 2008). After the introduction of bismuth (Bi) film electrode (Wang et al., 2000), Bi-based electrode has been studied in-depth and considered as an attractive alternative to mercury electrode because of its similar property in easy formation of alloys with heavy metals (Wang et al., 2000;Nedeltcheva et al., 2005;Wang, 2005;Saturno et al., 2011). More importantly, Bi-based electrode is much more environmental friendly than mercury electrode. Graphene (GR) is a planar two-dimensional (2D) honeycomblike lattice consists of sp2-hybridized carbon atoms monolayer. It has attracted considerable attention as electrode modifier because of its large specific surface area, outstanding electrical conductivity, and excellent electrochemical properties (Brownson and Banks, 2010;Gan and Hu, 2011;Han et al., 2014;Cinti and Arduini, 2017). The three-dimensional (3D) architecture of 2D graphene is an emerging graphene-based nanomaterial which provides a larger electroactive surface area and better opportunity for the loading of functional materials to construct electro-/bio-chemical sensors with excellent sensing performance (Baig and Saleh, 2018). 3D graphene which can be prepared by electrochemical methods has been proved to be a promising and outstanding electrode modifier and ideal platform for the combination of functional materials for electrochemical sensing (Dong et al., 2012;Yang et al., 2015;Baig and Saleh, 2018).
Acupuncture needle (ANE) which has been using for medical therapies originating from China is now a new emerging electrode substrate for the fabrication of novel electrochemical sensors with high sensitivity (Zhou et al., 2017;Han et al., 2018Han et al., , 2019. In recent years, the micro-needle electrode based on stainless steel ANE has been most widely adopted due to its advantages in flexibility, easy operation, and low price (Tang et al., 2015;Niu et al., 2018). The electrochemical sensing platform based on the micro-needle electrode exhibits much superior performance in the field of electrochemical analysis. Tang et al. (2017) used the unique needle shape of ANE to fabricate a microsensor for real-time monitoring of nitric oxide in acupoints of rats. Li et al. (2019) used the advantage of large specific surface area of ANE to prepare a copper microspheres and polyaniline film modified micro-needle electrode which has good performance for nitrate determination. Han et al. (2019) also used the large specific surface area of ANE and prepared a porous gold modified ANE with excellent performance for copper determination. So, considering the low price, non-toxicity, large specific surface area, and excellent performance of ANE, it was used in this work.
The main aim of this work was to fabricate a novel electrode for the determination of Pb in coastal waters. Based on this, a micro-needle electrode was constructed and functionalized with 3D reduced graphene oxide (3DrGO) and flower-like Bi nanosheets (F-BiNSs). The so-fabricated 3DrGO/F-BiNSs functionalized ANE (3DrGO/F-BiNSs/ANE) exhibited a good performance for the anodic stripping voltammetric determination of Pb 2+ . Additionally, the practical application of the novel functional micro-needle electrode for Pb 2+ determination in coastal water samples was conducted with satisfactory results.

Reagents and Apparatus
The stock standard solution of Pb 2+ (1000 mg L −1 ) was purchased from the National Research Centre for Certified Reference Materials (CRMs), Beijing, China. All the other reagents were analytical-grade chemicals and used without further purification. Acetate buffer solutions with pH ranging from 3.0 to 5.0 were prepared by 0.1 mol L −1 acetic acid and 0.1 mol L −1 sodium acetate. Deionized water (18.2 M cm specific resistance) obtained with a Pall Cascada laboratory water system was used throughout. Graphene oxide (GO) was provided by Nanjing XFNANO Materials Tech Co., Ltd. (Nanjing, China). Acupuncture needles (stainless steel, 0.25 × 60 mm) were provided by Suzhou Medical Appliance Factory Co., Ltd., China. Silicone rubber (NANDA no. 704) was obtained from Liyang Kangda Chemical Co., Ltd., China.
The morphology characterization and element analysis of the bare ANE, 3DrGO modified ANE (3DrGO/ANE) and 3DrGO/F-BiNSs/ANE were conducted on scanning electron microscopy (SEM, Hitachi S-4800 microscope, Japan) and energy dispersive X-ray spectroscopy (EDS, HORIBAEX-350, Japan), respectively. All the electrochemical experiments were performed on a CHI 660E Electrochemical Work Station (CH Instruments, Inc., Shanghai, China) using a conventional three-electrode system. The functional micro-needle electrode was used as the working electrode, with an Ag/AgCl electrode and platinum foil serving as reference and counter electrode, respectively. 797 VA computrace analyzer (Metrohm Ltd., Switzerland) was used for the validation of the values obtained with the 3DrGO/F-BiNSs/ANE. All potentials were measured with respect to the Ag/AgCl reference electrode.

Preparation of the 3DrGO/F-BiNSs/ANE
The fabrication process of the 3DrGO/F-BiNSs/ANE was shown in Figure 1. Before modification, the bare micro-needle electrode (ANE) was prepared according to our previous work with little modification . Simply, the body part of the ANE was covered with silicone rubber for sealing, leaving the needle tip (about 1 mm) as sensing surface and the needle handle as electrode wire. The modification of ANE with 3DrGO was conducted by cycling the potentials from 0.5 to −1.5 V in 0.5 mg L −1 GO solution for 30 circles with ANE as the working electrode (Baig and Saleh, 2018). The 3DrGO/F-BiNSs/ANE was prepared by electrodepositing at −0.6 V in 0.5 mmol L −1 Bi(NO 3 ) 3 solution for 60 s with 3DrGO/ANE as the working electrode. The functional micro-needle electrode was obtained eventually after the cleaning and drying processes at room temperature.

Electrochemical Analysis Procedures
Unless otherwise stated, 0.1 mol L −1 acetate buffer solution was adopted as the supporting electrolyte for the determination of Pb 2+ . Quantitative determination of Pb 2+ was performed with square wave voltammetry (SWV) from −0.65 to −0.4 V (−0.8 to −0.3 V for the determination of Pb in coastal water samples) by using the following parameters: accumulation potential of −0.75 V, accumulation time of 240 s, amplitude of 0.025 V, potential incremental of 0.004 V, frequency of 25 Hz, and quiet time of 2 s. After one measurement, a cleaning process was applied to the electrode at −0.4 V for 30 s with stirring. The standard addition method was adopted for the determination of Pb 2+ in coastal water samples. The electrochemical impedance spectroscopy (EIS) of different modified micro-needle electrodes was conducted in 5 mmol L −1 K 3 [Fe(CN) 6 ] (0.1 mol L −1 KCl) solution with the frequency ranging from 10 −2 to 10 5 Hz.

Stability, Repeatability and Selectivity
The stability of the functional micro-needle electrode was evaluated by determining 1 µmol L −1 Pb 2+ at the same 3DrGO/F-BiNSs/ANE for six measurements. The reproducibility was investigated by determining 1 µmol L −1 Pb 2+ at six independently fabricated 3DrGO/F-BiNSs/ANEs. The selectivity was evaluated by the influence of different potential foreign ions (K + , Na + , Ca 2+ , Mg 2+ , Cr 3+ , NO 3 − , SO 4 2− , Cl − , Co 2+ , Cd 2+ , Cu 2+ ) on the electrochemical response of Pb 2+ at the 3DrGO/F-BiNSs/ANE. Current responses of 1 µmol L −1 Pb 2+ were obtained after the addition of different foreign species into acetate buffer solution under the optimal conditions.

Preparation of Coastal Water Samples
The coastal water samples were collected from the Yuniao River Estuary (Shandong Province, China) at different locations. All the water samples were filtered with 0.45 µm membrane filters and kept in the fridge (4 • C) until determination. Before determination with standard addition method, the samples were adjusted to pH 3.5 by using 0.4 mol L −1 acetic acid solutions.

Characterization of the 3DrGO/F-BiNSs/ANE
The morphologies of different modified micro-needle electrodes were investigated by SEM (Figure 2). The SEM image of the so-prepared bare ANE and its low magnification image (the insert) were showed in Figure 2A. It can be seen that the surface of the bare ANE was very smooth which provided limited binding sites for the combination of functional nanomaterials. The diameter of the needle tip was around 5 µm as described in our previous publications (Han et al., 2018. Figure 2B showed the SEM image of the surface of 3DrGO modified ANE which exhibited an interconnected 3D network of rGO sheets. The morphology was consistent with the 3D graphene synthesized and reported in the previous publication (Cui et al., 2013). The 3D graphene network provided larger exposed surface area and more active sites which were helpful for the direct electron transfer and combination of functional nanomaterials. After the process of electrochemical deposition in Bi(NO 3 ) 3 solution, Bi nanosheets with flowerlike structure were distributed on the surface of 3DrGO (Figures 2C,D). From the SEM characterization, it can be concluded that all the 3DrGO and F-BiNSs could be modified on the micro-needle electrode by the electrodeposition method. The novel functional micro-needle electrode exhibited a good performance for the voltammetric determination of Pb 2+ as discussed below.
To investigate the elemental composition of different modified micro-needle electrodes, EDS was conducted (Figure 3). It can be seen clearly that Fe, Cr, Ni, C, and Si were the main elements of the stainless steel ANE. When compared with the bare ANE, the content percentage of C increased obviously in the 3DrGO/ANE, which might be caused by the presence of 3DrGO. After the electrochemical deposition of F-BiNSs on 3DrGO/ANE, the typical Bi peaks at about 2.42, 9.45, and 10.81 KeV appeared on the EDS pattern of 3DrGO/F-BiNSs/ANE, which proved the presence of F-BiNSs. It can be concluded from the results of SEM and EDS characterizations that the functional micro-needle electrode, 3DrGO/F-BiNSs/ANE, was successfully fabricated.
Cyclic voltammetry (CV) and linear sweep voltammetry (LSV) were used to investigate the electrochemical properties of the functional micro-needle electrode. The cyclic voltammograms of different electrodes were recorded from −0.4 to 0.6 V in acetate buffer solution ( Figure 4A). There was no peak could be observed in the cyclic voltammograms of bare ANE and 3DrGO/ANE. However, the background current of 3DrGO/ANE was much higher than that of bare ANE, which proved the excellent conductivity of 3DrGO. After the electrodeposition of F-BiNSs, the 3DrGO/F-BiNSs/ANE exhibited a sharp typical oxidation peak of Bi at 0.08 V. The results of LSV were similar with that of CV, and there was almost no difference between ANE and 3DrGO/ANE ( Figure 4B). However, the oxidation peak of Bi at 0.16 V appeared on the 3DrGO/F-BiNSs/ANE. So, the 3DrGO and F-BiNSs could be modified on the ANE by electrodeposition method and the F-BiNSs might be stripped out when the potential exceeded 0.08 V.
Electrochemical impedance spectroscopy was conducted in K 3 [Fe(CN) 6 ] solution to investigate the electron transfer capability of different micro-needle electrodes (Figure 5). It is known that the charge transfer resistance (Rct) can be reflected by the semicircle diameter of Nyquist plot at high frequency part. It can be observed that the semicircle diameter of 3DrGO/ANE was much smaller than that of bare ANE which reflected the excellent conductivity of 3DrGO. However, after the modification of F-BiNSs, the Rct became relatively larger than that of 3DrGO/ANE. It was obvious that the electro-conductivity of F-BiNSs was relatively weaker than that of 3DrGO. Despite its relatively weak conductivity, F-BiNSs  enhanced the electrochemical sensing performance of the micro-needle electrode for Pb 2+ as discussed below due to their excellent property in easy formation of alloys with heavy metals.

Electrochemical Response of Lead on the 3DrGO/F-BiNSs/ANE
The electrochemical responses of Pb 2+ on different micro-needle electrodes were also investigated by using SWV (Figure 6). The bare ANE and 3DrGO/ANE had no obvious current response to 1 µmol L −1 Pb 2+ . So, there was no obvious enhancement for Pb 2+ determination when 3DrGO was modified on the micro-needle electrode. However, it provided larger exposed surface area and more active sites for the combination of Bi nanomaterials which could enhance the electrochemical voltammetric responses of Pb 2+ significantly. As expected,  a large oxidation peak at about −0.54 V appeared on the voltammogram of 3DrGO/F-BiNSs/ANE. It was obvious that the 3DrGO/F-BiNSs/ANE showed an enhanced performance for the voltammetric determination of Pb 2+ due to the combining ability of F-BiNSs with heavy metals (Pb here) and the 3D structure and excellent conductivity of 3DrGO.

Optimization for Pb Response on the 3DrGO/F-BiNSs/ANE
The effect of pH value of the acetate buffer for the stripping voltammetric determination of Pb 2+ on the 3DrGO/F-BiNSs/ANE was investigated ( Figure 7A). Stripping peak current (Ip) was obtained for 1 µmol L −1 Pb 2+ determination with the pH value ranging from 3.0 to 5.0. It can be seen that the maximum oxidation Ip appeared at pH 3.5 and decreases when the pH increased from 3.5 to 5.0. As reported, the decrease of Ip at pH higher than 3.5 might be explained as the hydrolysis of Pb 2+ . As a result, a pH of 3.5 was selected and used for the voltammetric determination of Pb 2+ in this work.
It is known that the accumulation potential has significant effect on the stripping voltammetric responses of metal ions. The effect of accumulation potential on the Ip of 1 µmol L −1 Pb 2+ obtained with the 3DrGO/F-BiNSs/ANE was investigated with the potential varying from −0.55 to −0.85 V (Figure 7B). It can be seen that the Ip increased continuously with the potential from −0.55 to −0.85 V. More Pb 2+ could be reduced and accumulated on the electrode surface by the more negative potentials in this process, which led to the stronger current response. Though a more negative potential could increase the accumulation amount of Pb 2+ and improve the sensitivity, it also enhanced the noise current of the micro-needle electrode. So, −0.75 V was selected as the accumulation potential and used throughout the work.  The effect of accumulation time on the current response was also investigated with the time ranging from 30 to 360 s when the deposition potential was fixed at −0.75 V ( Figure 7C). It can be seen that although the Ip increased continuously with the accumulation time ranging from 30 to 360 s, the increasing trend slowed down after the time reaching to 240 s. So, in this work, 240 s was adopted as the accumulation time for the determination of Pb 2+ in coastal water samples. Additionally, a prolonged accumulation time could be selected for the determination of Pb 2+ with lower concentration.
Performance Evaluation of the 3DrGO/F-BiNSs/ANE Figure 8 showed the square wave voltammograms and the corresponding calibration curve of different concentrations of Pb 2+ on the 3DrGO/F-BiNSs/ANE under the optimal conditions. The Ip was linear with the concentration of Pb 2+ ranging from 40 to 600 nmol L −1 with the equation for linear regression Ip = 299.98 C + 2.95 (R 2 = 0.990). The detection limit (LOD) of the 3DrGO/F-BiNSs/ANE for the voltammetric determination of Pb 2+ was calculated as 12.5 nmol L −1 . It should be noted that the detection performance could be enhanced by adjusting the accumulation potential and time as described above. Additionally, the comparison of the functional micro-needle electrode with other modified electrodes for the determination of Pb 2+ was presented in Table 1. When compared with the modified electrodes developed previously, the newly fabricated 3DrGO/F-BiNSs/ANE showed a satisfactory performance for the voltammetric determination of Pb 2+ . Furthermore, the 3DrGO/F-BiNSs/ANE exhibited good stability, repeatability and selectivity for the voltammetric determination of Pb 2+ . The relative standard deviation (RSD) of six determinations of 1 µmol L −1 Pb 2+ at the same 3DrGO/F-BiNSs/ANE was calculated as 3.8%. The RSD of six independently fabricated 3DrGO/F-BiNSs/ANEs for the determination of 1 µmol L −1 Pb 2+ was 5.2%. The results of selectivity evaluation showed that 100-fold K + , Na + , Ca 2+ , Mg 2+ , Cr 3+ , NO 3 − , SO 4 2− , Cl − , 50-fold Zn 2+ , 20-fold Co 2+ , Cd 2+ , and 10-fold Cu 2+ had no obvious effect on the voltammetric determination of Pb 2+ (<5% current change). Therefore the functional micro-needle electrode might be a good choice for the determination of Pb 2+ .

Determination of Pb 2+ in Coastal Waters With the 3DrGO/F-BiNSs/ANE
The practical application of the functional micro-needle electrode for the determination of Pb 2+ in real coastal water samples was also evaluated. Figure 9 showed the typical linear regression and corresponding square wave voltammograms obtained after the successive addition of 0, 50, and 150 nmol L −1 Pb 2+ . The Pb 2+ concentration in this coastal water sample was calculated to be 47.3 nmol L −1 . The Pb 2+ concentrations of different coastal water samples and corresponding recoveries were presented in Table 2. For comparison, the coastal water samples were also determined with the common mercury electrode via Metrohm 797 VA computrace analyzer (Zhao et al., 2019). The results obtained by these two methods were consistent. The results showed that the 3DrGO/F-BiNSs/ANE fabricated here could be used for the voltammetric determination of Pb 2+ in fresh and brackish water samples.

CONCLUSION
This work described a new method for the determination of Pb in coastal waters with a cheap and non-toxic functional micro-needle electrode. The detection performance of the micro-needle electrode was significantly enhanced by the 3DrGO/F-BiNSs. The newly fabricated 3DrGO/F-BiNSs/ANE exhibited a good performance for the voltammetric determination of Pb 2+ . The practical application of the functional micro-needle electrode for Pb determination in coastal water samples was conducted. Furthermore, the distribution of Pb in coastal waters of different regions could be investigated with the functional micro-needle electrode in the future.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/supplementary material.

AUTHOR CONTRIBUTIONS
HH and DP designed the experiments and wrote the manuscript. YL, JW, and CW sampled the coastal waters. HH performed the experiments and analyses.

ACKNOWLEDGMENTS
We would like to thank Dr. Wenhai Wang for the SEM and EDS characterizations of the modified electrodes.