Journal of Hazardous Materials 283 (2015) 157–163
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Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Liquid-phase exfoliated graphene as highly-sensitive sensor for simultaneous determination of endocrine disruptors: Diethylstilbestrol and estradiol Lintong Hu a , Qin Cheng a , Danchao Chen b , Ming Ma b , Kangbing Wu a,∗ a Key Laboratory for Large-Format Battery Materials and System, Ministry of Education, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China b Ningbo Entry-exit Inspection and Quarantine Bureau of China, Ningbo 315012, China
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• A novel electrochemical sensor was developed for diethylstilbestrol and estradiol. • Graphene prepared by solvent exfoliation greatly enhances the detection sensitivity. • The newly-developed method has promising application and the accuracy is good.
a r t i c l e
i n f o
Article history: Received 21 May 2014 Received in revised form 1 August 2014 Accepted 24 August 2014 Available online 18 September 2014 Keywords: Endocrine disruptors Highly-sensitive detection Diethylstilbestrol Estradiol Exfoliated graphene Electrochemical sensor
a b s t r a c t It is quite important to develop convenient and rapid analytical methods for trace levels of endocrine disruptors because they heavily affect health and reproduction of humans and animals. Herein, graphene was easily prepared via one-step exfoliation using N-methyl-2-pyrrolidone as solvent, and then used to construct an electrochemical sensor for highly-sensitive detection of diethylstilbestrol (DES) and estradiol (E2). On the surface of prepared graphene film, two independent and greatly-increased oxidation waves were observed at 0.28 V and 0.49 V for DES and E2. The remarkable signal enlargements indicated that the detection sensitivity was improved significantly. The influences of pH value, amount of graphene and accumulation time on the oxidation signals of DES and E2 were discussed. As a result, a highly-sensitive and rapid electrochemical method was newly developed for simultaneous detection of DES and E2. The values of detection limit were evaluated to be 10.87 nM and 4.9 nM for DES and E2. Additionally, this new method was successfully used in lake water samples and the accuracy was satisfactory. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Endocrine disruptors, also called environmental estrogens, have attracted much attention of the scientific community and the general public. Endocrine disruptors are estrogen-like and/or
∗ Corresponding author. Tel.: +86 27 87543632; fax: +86 27 87543632. E-mail addresses: [email protected], [email protected] (K. Wu). http://dx.doi.org/10.1016/j.jhazmat.2014.08.067 0304-3894/© 2014 Elsevier B.V. All rights reserved.
antiandrogenic chemicals that can disrupt hormonal balance and result in developmental and reproductive abnormalities [1]. Additionally, new in vitro, in vivo, and epidemiological studies link human endocrine disruptor exposure with obesity, metabolic syndrome, type 2 diabetes, and cancer [2,3]. Generally, endocrine disruptors in the environment include natural estrogens and artificially synthesized estrogens [4]. Currently, diethylstilbestrol (DES) and estradiol (E2) have been considered as typical examples of synthetic and natural endocrine disruptors. Therefore, it is quite
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important to develop sensitive and convenient methods for the determination of DES and E2 in the environment. The widely-employed analytical methods for DES and E2 were high performance liquid chromatography (HPLC) [5,6] and liquid chromatography–mass spectrometry (LC–MS) [7,8]. Compared with HPLC and LC–MS, electrochemical detection displays some advantages such as portability, field-deployability, short analysis time, good handling convenience and low cost. From the chemical structures, we found that DES and E2 contain electrochemically-active group. So DES and E2 were also detected using electrochemical technique. For example, a carbon paste electrode (CPE) in the presence of cetylpyridine bromide [9], a -cyclodextrin functionalized reduced graphene oxide (RGO)modified glassy carbon electrode (GCE) [10], a conductive carbon black paste electrode [11], and a carbon nanotube/platinum nanoparticles-modified GCE [12] were used as electrochemical sensor for DES. In addition, various electrochemical methods were also developed for E2 detection using different sensing materials, such as RGO/dihexadecylphosphate composite film [13], molecularly imprinted membranes on Pt nanoparticles [14], poly (l-serine) film [15], carbon nanotubes/ionic liquids composite film [16], Pt nanoclusters/multi-walled carbon nanotubes [17], nano-Al2 O3 film [18] and carbon nanotube-Nafion composite film [19]. However, simultaneous detection of DES and E2 using electrochemical methods is very limited. The main objective of this work is to develop a highly-sensitive electrochemical method for simultaneous detection of DES and E2 using graphene-modified electrode. Thanks to large surface area, high catalytic activity and strong accumulation ability, graphene has obtained considerable attention in the field of electrochemical detection [20]. Among these studies, graphene was almost prepared through chemical exfoliation of graphite using strong oxidizing reagents, especially according to Hummer’s [21] or modified Hummer’s methods [22]. However, preparation of graphene through chemical oxidation has some intrinsic drawbacks, such as huge consumption of chemicals, complicated procedure and rigorous conditions. Compared with chemical oxidation strategy, liquid-phase exfoliation has been proven to be an effective, mild and simple approach to prepare graphene [23]. Herein, graphene nanosheets were easily obtained through onestep exfoliation of graphite powder in N-methyl-2-pyrrolidone (NMP). After that, the resulting graphene samples were dispersed into N,N-dimethylformamide (DMF), forming a stable suspension. By solvent evaporation, a liquid-phase exfoliated graphene filmmodified GCE was achieved. The electrochemical responses of DES and E2 were studied, and it was found that their oxidation waves were independent and the peak currents greatly increased on the surface of prepared graphene film. Undoubtedly, the developed graphene-modified electrode is quite qualified for the simultaneous detection of trace levels of DES and E2. Compared with the
reported electrochemical sensors that are listed in Table 1, we clearly found that the newly-developed sensor exhibited higher sensitivity and shorter analysis time.
2. Experimental 2.1. Reagents All chemicals were of analytical grade and used as received. DES and E2 were received from Sigma, and individually dissolved into ethanol to prepare 0.01 M standard solution. Graphite powder (spectral pure), NMP and DMF were purchased from Sinopharm Chemical Reagent Company (Shanghai, China). Ultrapure water (18.2 M) was obtained from a Milli-Q water purification system and used throughout.
2.2. Instruments Electrochemical measurements were performed on a CHI 830D electrochemical workstation (Chenhua Instrument, Shanghai, China) with a conventional three-electrode system. The working electrode was an exfoliated graphene-modified GCE, the reference electrode was a saturated calomel electrode (SCE), and the counter electrode was platinum wire. Scanning electron microscopy (SEM) characterization was conducted with a Quanta 200 microscope (FEI Company, Netherlands). Transmission electron microscopy (TEM) characterization was conducted with a JEM-2100 electron microscope (JEOL, Japan). Atomic Force Microscopy (AFM) characterization was conducted with a SPM-9700 scanning probe microscope (SHIMADZU, Japan).
2.3. Construction of graphene-based electrochemical sensor Firstly, exfoliated graphene was obtained via ultrasonic exfoliation of graphite powder in NMP solvent. In a typical preparation, 5.0 g graphite powder was added into 500.0 mL NMP, and then sonicated in a KQ-100B ultrasonicator (frequency: 40 kHz, powder: 100 W) for 48 h. After that, the resulting suspension was centrifugated, washed with ultrapure water and ethanol several times, and finally dried in vacuum at 60 ◦ C for 5 h. Secondly, 10.0 mg of obtained graphene samples were exactly weighed, and then added into 5.0 mL of DMF. After 30-min ultrasonication, a stable suspension with concentration of 2.0 mg mL−1 was achieved. Finally, GCE with diameter of 3 mm was polished with 0.05 m alumina slurry, and ultrasonically washed with ultrapure water to give a clean surface. After that, 3.0 L of graphene suspension was added on GCE surface, and then dried under an infrared lamp in air.
Table 1 Performance comparison of electrochemical sensors for DES and E2. Analyte
Sensing material
Detection limit
Time
Ref.
DES
Carbon paste/cetylpyridine bromide -Cyclodextrin functionalized RGO Conductive carbon black Carbon nanotube/Pt nanoparticles Exfoliated graphene RGO/dihexadecylphosphate Molecularly imprinted membranes Poly (L-serine) film Carbon nanotubes/ionic liquid Pt nano-clusters/carbon nanotubes Nano-Al2 O3 film Carbon nanotube-Nafion Exfoliated graphene
1 × 10−8 M 4 × 10−9 M 8 × 10−9 M 1.5 × 10−8 M 1.1 × 10−8 M 7.7 × 10−8 M 1.6 × 10−8 M 2 × 10−8 M 5 × 10−9 M 1.8 × 10−7 M 8 × 10−8 M 1 × 10−8 M 4.9 × 10−9 M
5 min 3.3 min 3 min 2 min 2 min 4 min 4 min 2 min 4.5 min 3 min 2 min 5 min 2 min
[9] [10] [11] [12] This work [13] [14] [15] [16] [17] [18] [19] This work
E2
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2.4. Analytical procedure Unless otherwise stated, 0.1 M phosphate buffer with pH of 7.0 was used as the supporting electrolyte for the detection of DES and E2. After 2-min accumulation at 0 V, the differential pulse voltammograms were recorded from 0.0 to 0.7 V, and the oxidation peak currents at 0.28 V and 0.49 V were measured. The pulse amplitude was 50 mV, the pulse width was 40 ms and the scan rate was 40 mV s−1 . 3. Results and discussion 3.1. Properties of the prepared graphene The structure of exfoliated graphene was determined by TEM, and the results were shown in Fig. 1. A number of transparent nanosheets were clearly observed, suggesting that graphite powder was successfully exfoliated into graphene nanosheets. In addition, the surface morphologies of GCE and exfoliated graphene-modified GCE were characterized using SEM and AFM. From Fig. 2A and B, we clearly found that the bare GCE surface was smooth and virtually featureless. After modification with exfoliated graphene, it was clearly observed that the GCE surface was coated by nanosheets (Fig. 2C). AFM measurements further revealed that the surface roughness of graphene-modified GCE increased greatly (Fig. 2D).
Fig. 1. TEM image of exfoliated graphene.
Undoubtedly, the existence of nanosheets and the increased surface roughness certainly enhance the response area and electrochemical reactivity. Fig. 3 compared the electrochemical responses of potassium ferricyanide (K3 [Fe(CN)6 ]) on the bare GCE and the graphenemodified GCE. From the comparison, we clearly found that the peak
Fig. 2. SEM and AFM images of GCE (A, B) and exfoliated graphene-modified GCE (C, D).
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Fig. 3. Cyclic voltammograms of 5 mM K3 [Fe(CN)6 ] in 1 M KCl on GCE (a) and exfoliated graphene-modified GCE (b). Scan rate = 100 mV s−1 .
currents increased obviously on the surface of graphene-modified GCE. According to Randles-Sevcik equation, higher peak currents indicate larger active electrode area. Moreover, the reduction peak shifted positively and the oxidation wave moved negatively on the graphene-modified GCE. The decreased peak potential separation reveals that modification of graphene nanosheets facilitates the electron transfer. In brief, the graphene-modified GCE exhibits larger surface area and higher electrochemical reactivity, compared with the bare GCE. 3.2. Signal enhancement of exfoliated graphene The electrochemical responses of DES and E2 were examined using differential pulse voltammetry (DPV), and the results were displayed in Fig. 4. In pH 7.0 phosphate buffer and after 2-min accumulation, two negligible oxidation waves were observed on the bare GCE surface (curve b). The poor oxidation signals indicate low response activities on the unmodified GCE. Interestingly, two welldefined and sensitive oxidation peaks appeared at 0.28 V and 0.49 V when using graphene-modified GCE (curve d). The remarkable oxidation signal improvements suggest that the prepared graphene
Fig. 4. DPV curves of GCE (a and b) and graphene-modified GCE (c and d) in pH 7.0 phosphate buffer (a and c) and in the presence of 500 nM DES and E2 (b and d). Inset plots: DPV curves 500 nM DES and E2 on GO-modified GCE (e) and RGO-modified GCE (f). Accumulation time: 2 min, amount of suspension: 3 L, pulse amplitude: 50 mV, pulse width: 40 ms, scan rate: 40 mV s−1 .
facilitates the electrochemical oxidation of DES and E2, and consequently enhances the detection sensitivity. Otherwise, the DPV responses of bare GCE and graphene-modified GCE in the absence of DES and E2 were studied. As shown in curves (a) and (c), they were featureless and no oxidation waves appeared, revealing that the observed oxidation peaks at 0.28 V and 0.49 V were caused by the oxidation of DES and E2. According to the published results [10,12,24], the oxidation of DES and E2 is attributed to the phenolic hydroxyl group. In addition, the oxidation behaviors of DES and E2 on the graphene oxides (GO)-modified GCE and the reduced graphene oxides (RGO)-modified GCE were also investigated. GO and RGO were prepared through chemical exfoliation methods [25]. As seen in curves (e) and (f), no oxidation waves were clearly observed on the surface of GO and RGO for DES and E2. Moreover, the background currents became very large. Compared with GO and RGO, graphene nanosheets prepared by liquid-phase exfoliation possess more global coverage of electrochemically reactive edge plane sites and defects, which in turn result in the increased electrochemical activity of the electrode [26,27]. As a result, the accumulation efficiency of DES and E2 was improved remarkably, and finally the oxidation peak currents enhanced greatly on the surface of exfoliated graphene. In conclusion, the comparisons of Fig. 4 clearly demonstrate that the exfoliated graphene-based sensor is more sensitive for the simultaneous detection of DES and E2.
3.3. Simultaneous detection of DES and E2 The oxidation behaviors of DES and E2 in 0.1 M phosphate buffer solutions with different pH values were studied to discuss the influences of pH value. As seen in Fig. 5A, the oxidation peak currents of DES and E2 on graphene-modified GCE increased gradually with pH value from 5.7 to 7.0, and then gradually decreased with further improving pH value to 8.0. To obtain high sensitivity, pH 7.0 phosphate buffer was used for the determination of DES and E2. The effects of amount of graphene suspension on the oxidation signals of DES and E2 were also investigated, and the results were shown in Fig. 5B. It was found that the oxidation peak currents of DES and E2 enhanced remarkably when improving the amount of graphene suspension from 0 to 3 L. During this period, the increased graphene on GCE surface obviously enhances the accumulation efficiency of DES and E2. As a result, the oxidation signals of DES and E2 were greatly improved. As further increasing the amount to 5 L, the oxidation peak currents of DES and E2 changed slightly, indicative of a saturation status. For higher sensitivity and shorter time for solvent evaporation, 3 L of graphene suspension was employed for GCE modification. Accumulation is a simple and effective approach to further improve the detection sensitivity, and it mainly contains two parameters: potential and time. For handling convenience, the accumulation of DES and E2 on graphene surface was conducted at the initial potential. Fig. 5C illustrates the variation of oxidation signals with the accumulation potentials. We clearly found that the oxidation peak currents of DES and E2 changed slightly when the initial potential altered from −0.30 V to 0.10 V, suggesting that the influences of accumulation potential is limited. For better shape of oxidation wave, the initial potential was fixed at 0.0 V. Fig. 5D demonstrates the influences of accumulation time on the oxidation peak currents of DES and E2. By extending the accumulation time from 0 to 2 min, the oxidation peak currents of DES and E2 increased significantly, indicating that accumulation is highly efficient to improve the detection sensitivity. When further improving the accumulation time to 5 min, the oxidation peak currents of E2 increased slightly, while the oxidation peak currents of DES almost kept unchanged. Considering sensitivity and efficiency, 2-min accumulation was used.
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Fig. 5. Effects of pH value (A), amount of graphene suspension (B), accumulation potential (C) and accumulation time (D) on the oxidation peak currents of 500 nM DES (a) and E2 (b). Other conditions were the same as in Fig. 4. Error bar represents the standard deviation of triple measurements.
The electrochemical responses of coexistence of DES and E2 on graphene-modified GCE were examined using DPV in pH 7.0 phosphate buffer. Fig. 6A shows the DPV curves of DES in the presence of E2 with different concentrations. It was found that the oxidation signals of DES almost kept unchanged when the concentration of E2 increased from 10 nM to 1.5 M. A similar experiment was also carried out for DES, and the results were shown in Fig. 6B. When improving the concentration of DES from 25 nM to 3 M, the oxidation peak currents of E2 kept stable. Therefore, the oxidation of DES and E2 on graphene-modified GCE was independent, and their mutual interference was very slight. Moreover, the calibration curves for E2 and DES were given in Fig. 6C and D. For E2, the oxidation signals increased linearly with its concentration (C) over the ranger from 10 nM to 1.5 M, and the linear regression equation was: Ip = 0.00443C (Ip in A, C in nM, R = 0.999). As to DES, the linear ranger was from 25 nM to 3 M, obeying the following equation: Ip = 0.00165 C (Ip in A, C in nM, R = 0.999). Based on three signal-to-noise ratio, the detection limits were calculated to be 4.9 nM and 10.87 nM for E2 and DES. In this work, we found that the graphene-modified GCE was unqualified for successive measurements because the oxidation peak currents of DES and E2 decreased continuously. Thus, it was employed for single measurement. The reproducibility between multiple graphene-modified GCEs was then tested using 500 nM DES and E2. The relative standard deviation (RSD) was 5.4% (DES) and 3.2% (E2) for ten graphene-modified GCEs, indicative of excellent fabrication reproducibility and detection precision. The potential interferences for the determination of DES and E2 were studied. Under the optimized conditions, the oxidation peak currents of DES and E2 were individually measured in the presence of interferents with different concentrations, and the peak current
change was then checked. No influences on the detection of 500 nM DES and E2 were found after addition of 0.15 mM Zn2+ , Cd2+ and Mn2+ , 0.1 mM Cu2+ , Pb2+ and uric acid, 0.05 mM ascorbic acid, and 0.01 mM Hg2+ and dopamine (peak current changes < 10%). 3.4. Practical application In order to testify its practical application, this sensor was employed to detect DES and E2 in different water samples. The samples were collected from different lakes in Wuhan City, and then filtered before detection. Upon addition of 5.0 mL sample solution into 5.0 mL pH 7.0 phosphate buffer, the DPV curves were recorded from 0 to 0.7 V after 2-min accumulation. Each sample was determined by three times, and the RSD was lower than 5%, revealing good precision. The contents of DES and E2 were obtained using standard additional method, and the results were listed in Table 2. The value of recovery was in the range from 99.0% to 104.4%, indicating that the newly-developed method is accurate and feasible.
Table 2 Detection of DES and E2 in different lake water samples. No. 1 2 3 4
Analyte
Measured (M)
Added (M)
Found (M)
Recovery (%)
DES E2 DES E2 DES E2 DES E2
4.913 × 10−8 2.458 × 10−8 9.800 × 10−8 4.800 × 10−8 1.595 × 10−7 1.050 × 10−7 1.931 × 10−7 1.522 × 10−7
5.00 × 10−8 2.50 × 10−8 1.00 × 10−7 5.00 × 10−8 1.50 × 10−7 1.00 × 10−7 2.00 × 10−7 1.50 × 10−7
9.864 × 10−8 5.043 × 10−8 2.001 × 10−7 1.002 × 10−7 3.146 × 10−7 2.080 × 10−7 3.947 × 10−7 3.058 × 10−7
99.0 103.4 102.1 104.4 103.4 103.0 100.8 102.4
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Fig. 6. (A) DPV curves of 500 nM DES in the presence of E2 with concentrations of 10 nM (a), 25 nM (b), 50 nM (c), 100 nM (d), 250 M (e), 500 nM (f), 1 M (g), 1.5 M (h). (B) DPV curves of 500 nM E2 in the presence of DES with concentrations of 25 nM (a), 50 nM (b), 100 nM (c), 500 nM (d), 1 M (e), 2 M (f), 3 M (g). (C) Calibration curve of E2. (D) Calibration curve of DES.
4. Conclusion Graphene was conveniently prepared via one-step solvent exfoliation, and then used as sensing material to greatly improve the oxidation signals of DES and E2. On the surface of graphene film, two independent oxidation waves appeared, and the peak currents enhanced greatly. So a novel electrochemical sensor with high sensitivity, short analysis time, good accuracy and promising application was successfully developed for the simultaneous detection of DES and E2. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 21375041 & 61071052), the National Basic Research Program of China (973 Program, No. 2015CB352100), the Program for New Century Excellent Talents in University (NCET-110187), and the Research foundation of State General Administration of The People’s Republic of China for Quality Supervision and Inspection and Quarantine (2013IK210). The Center of Analysis and Testing of Huazhong University of Science and Technology was acknowledged for its help in the SEM and AFM observation. References [1] S.C. Sikka, R. Wang, Endocrine disruptors and estrogenic effects on male reproductive axis, Asian J. Androl. 10 (2008) 134–145. [2] A.M. Soto, C. Sonnenschein, Environmental causes of cancer: endocrine disruptors as carcinogens, Nat. Rev. Endocrinol. 6 (2010) 363–370.
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Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Liquid-phase exfoliated graphene as highly-sensitive sensor for simultaneous determination of endocrine disruptors: Diethylstilbestrol and estradiol Lintong Hu a , Qin Cheng a , Danchao Chen b , Ming Ma b , Kangbing Wu a,∗ a Key Laboratory for Large-Format Battery Materials and System, Ministry of Education, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China b Ningbo Entry-exit Inspection and Quarantine Bureau of China, Ningbo 315012, China
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• A novel electrochemical sensor was developed for diethylstilbestrol and estradiol. • Graphene prepared by solvent exfoliation greatly enhances the detection sensitivity. • The newly-developed method has promising application and the accuracy is good.
a r t i c l e
i n f o
Article history: Received 21 May 2014 Received in revised form 1 August 2014 Accepted 24 August 2014 Available online 18 September 2014 Keywords: Endocrine disruptors Highly-sensitive detection Diethylstilbestrol Estradiol Exfoliated graphene Electrochemical sensor
a b s t r a c t It is quite important to develop convenient and rapid analytical methods for trace levels of endocrine disruptors because they heavily affect health and reproduction of humans and animals. Herein, graphene was easily prepared via one-step exfoliation using N-methyl-2-pyrrolidone as solvent, and then used to construct an electrochemical sensor for highly-sensitive detection of diethylstilbestrol (DES) and estradiol (E2). On the surface of prepared graphene film, two independent and greatly-increased oxidation waves were observed at 0.28 V and 0.49 V for DES and E2. The remarkable signal enlargements indicated that the detection sensitivity was improved significantly. The influences of pH value, amount of graphene and accumulation time on the oxidation signals of DES and E2 were discussed. As a result, a highly-sensitive and rapid electrochemical method was newly developed for simultaneous detection of DES and E2. The values of detection limit were evaluated to be 10.87 nM and 4.9 nM for DES and E2. Additionally, this new method was successfully used in lake water samples and the accuracy was satisfactory. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Endocrine disruptors, also called environmental estrogens, have attracted much attention of the scientific community and the general public. Endocrine disruptors are estrogen-like and/or
∗ Corresponding author. Tel.: +86 27 87543632; fax: +86 27 87543632. E-mail addresses: [email protected], [email protected] (K. Wu). http://dx.doi.org/10.1016/j.jhazmat.2014.08.067 0304-3894/© 2014 Elsevier B.V. All rights reserved.
antiandrogenic chemicals that can disrupt hormonal balance and result in developmental and reproductive abnormalities [1]. Additionally, new in vitro, in vivo, and epidemiological studies link human endocrine disruptor exposure with obesity, metabolic syndrome, type 2 diabetes, and cancer [2,3]. Generally, endocrine disruptors in the environment include natural estrogens and artificially synthesized estrogens [4]. Currently, diethylstilbestrol (DES) and estradiol (E2) have been considered as typical examples of synthetic and natural endocrine disruptors. Therefore, it is quite
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important to develop sensitive and convenient methods for the determination of DES and E2 in the environment. The widely-employed analytical methods for DES and E2 were high performance liquid chromatography (HPLC) [5,6] and liquid chromatography–mass spectrometry (LC–MS) [7,8]. Compared with HPLC and LC–MS, electrochemical detection displays some advantages such as portability, field-deployability, short analysis time, good handling convenience and low cost. From the chemical structures, we found that DES and E2 contain electrochemically-active group. So DES and E2 were also detected using electrochemical technique. For example, a carbon paste electrode (CPE) in the presence of cetylpyridine bromide [9], a -cyclodextrin functionalized reduced graphene oxide (RGO)modified glassy carbon electrode (GCE) [10], a conductive carbon black paste electrode [11], and a carbon nanotube/platinum nanoparticles-modified GCE [12] were used as electrochemical sensor for DES. In addition, various electrochemical methods were also developed for E2 detection using different sensing materials, such as RGO/dihexadecylphosphate composite film [13], molecularly imprinted membranes on Pt nanoparticles [14], poly (l-serine) film [15], carbon nanotubes/ionic liquids composite film [16], Pt nanoclusters/multi-walled carbon nanotubes [17], nano-Al2 O3 film [18] and carbon nanotube-Nafion composite film [19]. However, simultaneous detection of DES and E2 using electrochemical methods is very limited. The main objective of this work is to develop a highly-sensitive electrochemical method for simultaneous detection of DES and E2 using graphene-modified electrode. Thanks to large surface area, high catalytic activity and strong accumulation ability, graphene has obtained considerable attention in the field of electrochemical detection [20]. Among these studies, graphene was almost prepared through chemical exfoliation of graphite using strong oxidizing reagents, especially according to Hummer’s [21] or modified Hummer’s methods [22]. However, preparation of graphene through chemical oxidation has some intrinsic drawbacks, such as huge consumption of chemicals, complicated procedure and rigorous conditions. Compared with chemical oxidation strategy, liquid-phase exfoliation has been proven to be an effective, mild and simple approach to prepare graphene [23]. Herein, graphene nanosheets were easily obtained through onestep exfoliation of graphite powder in N-methyl-2-pyrrolidone (NMP). After that, the resulting graphene samples were dispersed into N,N-dimethylformamide (DMF), forming a stable suspension. By solvent evaporation, a liquid-phase exfoliated graphene filmmodified GCE was achieved. The electrochemical responses of DES and E2 were studied, and it was found that their oxidation waves were independent and the peak currents greatly increased on the surface of prepared graphene film. Undoubtedly, the developed graphene-modified electrode is quite qualified for the simultaneous detection of trace levels of DES and E2. Compared with the
reported electrochemical sensors that are listed in Table 1, we clearly found that the newly-developed sensor exhibited higher sensitivity and shorter analysis time.
2. Experimental 2.1. Reagents All chemicals were of analytical grade and used as received. DES and E2 were received from Sigma, and individually dissolved into ethanol to prepare 0.01 M standard solution. Graphite powder (spectral pure), NMP and DMF were purchased from Sinopharm Chemical Reagent Company (Shanghai, China). Ultrapure water (18.2 M) was obtained from a Milli-Q water purification system and used throughout.
2.2. Instruments Electrochemical measurements were performed on a CHI 830D electrochemical workstation (Chenhua Instrument, Shanghai, China) with a conventional three-electrode system. The working electrode was an exfoliated graphene-modified GCE, the reference electrode was a saturated calomel electrode (SCE), and the counter electrode was platinum wire. Scanning electron microscopy (SEM) characterization was conducted with a Quanta 200 microscope (FEI Company, Netherlands). Transmission electron microscopy (TEM) characterization was conducted with a JEM-2100 electron microscope (JEOL, Japan). Atomic Force Microscopy (AFM) characterization was conducted with a SPM-9700 scanning probe microscope (SHIMADZU, Japan).
2.3. Construction of graphene-based electrochemical sensor Firstly, exfoliated graphene was obtained via ultrasonic exfoliation of graphite powder in NMP solvent. In a typical preparation, 5.0 g graphite powder was added into 500.0 mL NMP, and then sonicated in a KQ-100B ultrasonicator (frequency: 40 kHz, powder: 100 W) for 48 h. After that, the resulting suspension was centrifugated, washed with ultrapure water and ethanol several times, and finally dried in vacuum at 60 ◦ C for 5 h. Secondly, 10.0 mg of obtained graphene samples were exactly weighed, and then added into 5.0 mL of DMF. After 30-min ultrasonication, a stable suspension with concentration of 2.0 mg mL−1 was achieved. Finally, GCE with diameter of 3 mm was polished with 0.05 m alumina slurry, and ultrasonically washed with ultrapure water to give a clean surface. After that, 3.0 L of graphene suspension was added on GCE surface, and then dried under an infrared lamp in air.
Table 1 Performance comparison of electrochemical sensors for DES and E2. Analyte
Sensing material
Detection limit
Time
Ref.
DES
Carbon paste/cetylpyridine bromide -Cyclodextrin functionalized RGO Conductive carbon black Carbon nanotube/Pt nanoparticles Exfoliated graphene RGO/dihexadecylphosphate Molecularly imprinted membranes Poly (L-serine) film Carbon nanotubes/ionic liquid Pt nano-clusters/carbon nanotubes Nano-Al2 O3 film Carbon nanotube-Nafion Exfoliated graphene
1 × 10−8 M 4 × 10−9 M 8 × 10−9 M 1.5 × 10−8 M 1.1 × 10−8 M 7.7 × 10−8 M 1.6 × 10−8 M 2 × 10−8 M 5 × 10−9 M 1.8 × 10−7 M 8 × 10−8 M 1 × 10−8 M 4.9 × 10−9 M
5 min 3.3 min 3 min 2 min 2 min 4 min 4 min 2 min 4.5 min 3 min 2 min 5 min 2 min
[9] [10] [11] [12] This work [13] [14] [15] [16] [17] [18] [19] This work
E2
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2.4. Analytical procedure Unless otherwise stated, 0.1 M phosphate buffer with pH of 7.0 was used as the supporting electrolyte for the detection of DES and E2. After 2-min accumulation at 0 V, the differential pulse voltammograms were recorded from 0.0 to 0.7 V, and the oxidation peak currents at 0.28 V and 0.49 V were measured. The pulse amplitude was 50 mV, the pulse width was 40 ms and the scan rate was 40 mV s−1 . 3. Results and discussion 3.1. Properties of the prepared graphene The structure of exfoliated graphene was determined by TEM, and the results were shown in Fig. 1. A number of transparent nanosheets were clearly observed, suggesting that graphite powder was successfully exfoliated into graphene nanosheets. In addition, the surface morphologies of GCE and exfoliated graphene-modified GCE were characterized using SEM and AFM. From Fig. 2A and B, we clearly found that the bare GCE surface was smooth and virtually featureless. After modification with exfoliated graphene, it was clearly observed that the GCE surface was coated by nanosheets (Fig. 2C). AFM measurements further revealed that the surface roughness of graphene-modified GCE increased greatly (Fig. 2D).
Fig. 1. TEM image of exfoliated graphene.
Undoubtedly, the existence of nanosheets and the increased surface roughness certainly enhance the response area and electrochemical reactivity. Fig. 3 compared the electrochemical responses of potassium ferricyanide (K3 [Fe(CN)6 ]) on the bare GCE and the graphenemodified GCE. From the comparison, we clearly found that the peak
Fig. 2. SEM and AFM images of GCE (A, B) and exfoliated graphene-modified GCE (C, D).
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Fig. 3. Cyclic voltammograms of 5 mM K3 [Fe(CN)6 ] in 1 M KCl on GCE (a) and exfoliated graphene-modified GCE (b). Scan rate = 100 mV s−1 .
currents increased obviously on the surface of graphene-modified GCE. According to Randles-Sevcik equation, higher peak currents indicate larger active electrode area. Moreover, the reduction peak shifted positively and the oxidation wave moved negatively on the graphene-modified GCE. The decreased peak potential separation reveals that modification of graphene nanosheets facilitates the electron transfer. In brief, the graphene-modified GCE exhibits larger surface area and higher electrochemical reactivity, compared with the bare GCE. 3.2. Signal enhancement of exfoliated graphene The electrochemical responses of DES and E2 were examined using differential pulse voltammetry (DPV), and the results were displayed in Fig. 4. In pH 7.0 phosphate buffer and after 2-min accumulation, two negligible oxidation waves were observed on the bare GCE surface (curve b). The poor oxidation signals indicate low response activities on the unmodified GCE. Interestingly, two welldefined and sensitive oxidation peaks appeared at 0.28 V and 0.49 V when using graphene-modified GCE (curve d). The remarkable oxidation signal improvements suggest that the prepared graphene
Fig. 4. DPV curves of GCE (a and b) and graphene-modified GCE (c and d) in pH 7.0 phosphate buffer (a and c) and in the presence of 500 nM DES and E2 (b and d). Inset plots: DPV curves 500 nM DES and E2 on GO-modified GCE (e) and RGO-modified GCE (f). Accumulation time: 2 min, amount of suspension: 3 L, pulse amplitude: 50 mV, pulse width: 40 ms, scan rate: 40 mV s−1 .
facilitates the electrochemical oxidation of DES and E2, and consequently enhances the detection sensitivity. Otherwise, the DPV responses of bare GCE and graphene-modified GCE in the absence of DES and E2 were studied. As shown in curves (a) and (c), they were featureless and no oxidation waves appeared, revealing that the observed oxidation peaks at 0.28 V and 0.49 V were caused by the oxidation of DES and E2. According to the published results [10,12,24], the oxidation of DES and E2 is attributed to the phenolic hydroxyl group. In addition, the oxidation behaviors of DES and E2 on the graphene oxides (GO)-modified GCE and the reduced graphene oxides (RGO)-modified GCE were also investigated. GO and RGO were prepared through chemical exfoliation methods [25]. As seen in curves (e) and (f), no oxidation waves were clearly observed on the surface of GO and RGO for DES and E2. Moreover, the background currents became very large. Compared with GO and RGO, graphene nanosheets prepared by liquid-phase exfoliation possess more global coverage of electrochemically reactive edge plane sites and defects, which in turn result in the increased electrochemical activity of the electrode [26,27]. As a result, the accumulation efficiency of DES and E2 was improved remarkably, and finally the oxidation peak currents enhanced greatly on the surface of exfoliated graphene. In conclusion, the comparisons of Fig. 4 clearly demonstrate that the exfoliated graphene-based sensor is more sensitive for the simultaneous detection of DES and E2.
3.3. Simultaneous detection of DES and E2 The oxidation behaviors of DES and E2 in 0.1 M phosphate buffer solutions with different pH values were studied to discuss the influences of pH value. As seen in Fig. 5A, the oxidation peak currents of DES and E2 on graphene-modified GCE increased gradually with pH value from 5.7 to 7.0, and then gradually decreased with further improving pH value to 8.0. To obtain high sensitivity, pH 7.0 phosphate buffer was used for the determination of DES and E2. The effects of amount of graphene suspension on the oxidation signals of DES and E2 were also investigated, and the results were shown in Fig. 5B. It was found that the oxidation peak currents of DES and E2 enhanced remarkably when improving the amount of graphene suspension from 0 to 3 L. During this period, the increased graphene on GCE surface obviously enhances the accumulation efficiency of DES and E2. As a result, the oxidation signals of DES and E2 were greatly improved. As further increasing the amount to 5 L, the oxidation peak currents of DES and E2 changed slightly, indicative of a saturation status. For higher sensitivity and shorter time for solvent evaporation, 3 L of graphene suspension was employed for GCE modification. Accumulation is a simple and effective approach to further improve the detection sensitivity, and it mainly contains two parameters: potential and time. For handling convenience, the accumulation of DES and E2 on graphene surface was conducted at the initial potential. Fig. 5C illustrates the variation of oxidation signals with the accumulation potentials. We clearly found that the oxidation peak currents of DES and E2 changed slightly when the initial potential altered from −0.30 V to 0.10 V, suggesting that the influences of accumulation potential is limited. For better shape of oxidation wave, the initial potential was fixed at 0.0 V. Fig. 5D demonstrates the influences of accumulation time on the oxidation peak currents of DES and E2. By extending the accumulation time from 0 to 2 min, the oxidation peak currents of DES and E2 increased significantly, indicating that accumulation is highly efficient to improve the detection sensitivity. When further improving the accumulation time to 5 min, the oxidation peak currents of E2 increased slightly, while the oxidation peak currents of DES almost kept unchanged. Considering sensitivity and efficiency, 2-min accumulation was used.
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Fig. 5. Effects of pH value (A), amount of graphene suspension (B), accumulation potential (C) and accumulation time (D) on the oxidation peak currents of 500 nM DES (a) and E2 (b). Other conditions were the same as in Fig. 4. Error bar represents the standard deviation of triple measurements.
The electrochemical responses of coexistence of DES and E2 on graphene-modified GCE were examined using DPV in pH 7.0 phosphate buffer. Fig. 6A shows the DPV curves of DES in the presence of E2 with different concentrations. It was found that the oxidation signals of DES almost kept unchanged when the concentration of E2 increased from 10 nM to 1.5 M. A similar experiment was also carried out for DES, and the results were shown in Fig. 6B. When improving the concentration of DES from 25 nM to 3 M, the oxidation peak currents of E2 kept stable. Therefore, the oxidation of DES and E2 on graphene-modified GCE was independent, and their mutual interference was very slight. Moreover, the calibration curves for E2 and DES were given in Fig. 6C and D. For E2, the oxidation signals increased linearly with its concentration (C) over the ranger from 10 nM to 1.5 M, and the linear regression equation was: Ip = 0.00443C (Ip in A, C in nM, R = 0.999). As to DES, the linear ranger was from 25 nM to 3 M, obeying the following equation: Ip = 0.00165 C (Ip in A, C in nM, R = 0.999). Based on three signal-to-noise ratio, the detection limits were calculated to be 4.9 nM and 10.87 nM for E2 and DES. In this work, we found that the graphene-modified GCE was unqualified for successive measurements because the oxidation peak currents of DES and E2 decreased continuously. Thus, it was employed for single measurement. The reproducibility between multiple graphene-modified GCEs was then tested using 500 nM DES and E2. The relative standard deviation (RSD) was 5.4% (DES) and 3.2% (E2) for ten graphene-modified GCEs, indicative of excellent fabrication reproducibility and detection precision. The potential interferences for the determination of DES and E2 were studied. Under the optimized conditions, the oxidation peak currents of DES and E2 were individually measured in the presence of interferents with different concentrations, and the peak current
change was then checked. No influences on the detection of 500 nM DES and E2 were found after addition of 0.15 mM Zn2+ , Cd2+ and Mn2+ , 0.1 mM Cu2+ , Pb2+ and uric acid, 0.05 mM ascorbic acid, and 0.01 mM Hg2+ and dopamine (peak current changes < 10%). 3.4. Practical application In order to testify its practical application, this sensor was employed to detect DES and E2 in different water samples. The samples were collected from different lakes in Wuhan City, and then filtered before detection. Upon addition of 5.0 mL sample solution into 5.0 mL pH 7.0 phosphate buffer, the DPV curves were recorded from 0 to 0.7 V after 2-min accumulation. Each sample was determined by three times, and the RSD was lower than 5%, revealing good precision. The contents of DES and E2 were obtained using standard additional method, and the results were listed in Table 2. The value of recovery was in the range from 99.0% to 104.4%, indicating that the newly-developed method is accurate and feasible.
Table 2 Detection of DES and E2 in different lake water samples. No. 1 2 3 4
Analyte
Measured (M)
Added (M)
Found (M)
Recovery (%)
DES E2 DES E2 DES E2 DES E2
4.913 × 10−8 2.458 × 10−8 9.800 × 10−8 4.800 × 10−8 1.595 × 10−7 1.050 × 10−7 1.931 × 10−7 1.522 × 10−7
5.00 × 10−8 2.50 × 10−8 1.00 × 10−7 5.00 × 10−8 1.50 × 10−7 1.00 × 10−7 2.00 × 10−7 1.50 × 10−7
9.864 × 10−8 5.043 × 10−8 2.001 × 10−7 1.002 × 10−7 3.146 × 10−7 2.080 × 10−7 3.947 × 10−7 3.058 × 10−7
99.0 103.4 102.1 104.4 103.4 103.0 100.8 102.4
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Fig. 6. (A) DPV curves of 500 nM DES in the presence of E2 with concentrations of 10 nM (a), 25 nM (b), 50 nM (c), 100 nM (d), 250 M (e), 500 nM (f), 1 M (g), 1.5 M (h). (B) DPV curves of 500 nM E2 in the presence of DES with concentrations of 25 nM (a), 50 nM (b), 100 nM (c), 500 nM (d), 1 M (e), 2 M (f), 3 M (g). (C) Calibration curve of E2. (D) Calibration curve of DES.
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