Materials Science and Engineering C 58 (2016) 1098–1104
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Voltammetric studies of Azathioprine on the surface of graphite electrode modified with graphene nanosheets decorated with Ag nanoparticles Elham Asadian a, Azam Iraji zad a,b, Saeed Shahrokhian a,c,⁎ a b c
Institute for Nanoscience and Nanotechnology (INST), Sharif University of Technology, Tehran, Iran Department of Physics, Sharif University of Technology, Tehran 14588-89694, Iran Department of Chemistry, Sharif University of Technology, Tehran 11155-9516, Iran
a r t i c l e
i n f o
Article history: Received 22 May 2015 Received in revised form 31 July 2015 Accepted 7 September 2015 Available online 12 September 2015 Keywords: Graphene nanosheets Ag Nanoparticles Modified electrode Azathioprine Voltammetry
a b s t r a c t By using graphene nanosheets decorated with Ag nanoparticles (AgNPs-G) as an effective approach for the surface modification of pyrolytic graphite electrode (PGE), a sensing platform was fabricated for the sensitive voltammetric determination of Azathioprine (Aza). The prepared AgNPs-G nanosheets were characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), UV–vis and Raman spectroscopy techniques. The electrochemical behavior of Aza was investigated by means of cyclic voltammetry. Comparing to the bare PGE, a remarkable enhancement was observed in the response characteristics of Aza on the surface of the modified electrode (AgNPs-G/PGE) as well as a noticeable decrease in its reduction overpotential. These results can be attributed to the incredible enlargement in the microscopic surface area of the electrode due to the presence of graphene nanosheets together with strong adsorption of Aza on its surface. The effect of experimental parameters such as accumulation time, the amount of modifier suspension and pH of the supporting electrolyte were also optimized toward obtaining the maximum sensitivity. Under the optimum conditions, the calibration curve studies demonstrated that the peak current increased linearly with Aza concentrations in the range of 7 × 10−7 to 1 × 10−4 mol L−1 with the detection limit of 68 nM. Further experiments revealed that the modified electrode can be successfully applied for the accurate determination of Aza in pharmaceutical preparations. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Azathioprine (Aza) (Fig. 1), belongs to the chemical class of purine analogues, is an immunosuppressive drug used in organ transplantation (usually kidney or liver) along with other medications to prevent new organ transplant immunological rejection. It is also used to treat an array of autoimmune diseases including rheumatoid arthritis [1]. Aza is a prodrug (a precursor of a drug), which is converted in the body to its active form called mercaptopurine and acts by decreasing the effects of certain cells in the body's immune system [2]. However, long term use of Aza increases the risk of developing certain types of cancer such as leukemia, lymphoma and skin cancer. Hence careful and routine monitoring of Aza's concentration is of great importance in clinical practices. A brief literature review reveals that various analytical techniques such as high performance liquid chromatography (HPLC) [3, 4], 1HNMR spectroscopy [5] and spectrophotometry [6] have been used for the determination of Aza in pharmaceutical formulations and biological fluids. Although these methods provide accurate measurements, they ⁎ Corresponding author at: Institute for Nanoscience and Nanotechnology (INST), Sharif University of Technology, Tehran, Iran. E-mail address: [email protected] (S. Shahrokhian).
http://dx.doi.org/10.1016/j.msec.2015.09.022 0928-4931/© 2015 Elsevier B.V. All rights reserved.
suffer from some disadvantages such as expensive instrumentations, tedious sample preparations, time consuming and low sensitivity and/or selectivity that make them inappropriate for regular analysis. Regarding to these issues a precise and accurate method for routine assays is of great importance. Electrochemical techniques come up with numerous advantages including low cost, simplicity, short analysis time and sensitivity. However electro-reduction of Aza at the surface of bare glassy carbon or pyrolytic graphite electrodes only shows a very weak and broad peak, revealing high overpotential values in its electrode process [7, 8]. One way to address this issue and improve the slow electron transfer kinetics as well as increase the active electrode surface area is to modify the electrode surface with various modifiers. From this point of view, nanomaterials such as nanotubes [9, 10], nanoparticles [11, 12], nanosheets [13–15] and so on are promising candidates. Graphene, a 2D one atom thick carbon nanosheet, have attracted a great deal of attention since its discovery in 2004 due to its outstanding properties: high electrical conductivity, large specific surface area (theoretical value ≈ 2630 m2 g−1), thermal and chemical stability and extraordinary mechanical strength [16]. All these properties together along with its 2D structure make graphene pioneer in various applications including energy storage devices (supercapacitors [17], batteries [18]), solar cells [19] and sensors [20–22]. Moreover, decorating graphene with other nanomaterials such as nanorods or nanoparticles provides
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NPs-G/modified PGE was prepared by casting a desired volume (20 μL in optimum condition) of the above mentioned AgNPs-G suspension on the PGE surface using a micropipette and left to dry at room temperature. Before the voltammetric measurements, the modified electrode was cycled between 0 and −1 V (scan rate 100 mV s−1) in 0.1 M phosphate buffer solution (pH 6.0) for several times until acquiring a stable background. 3. Results and discussion Fig. 1. Molecular structure of Azathioprine.
prominent properties, due to the synergetic effect between them [23–25]. Nanoparticles (NPs), usually exhibit size-related properties that differ significantly from those observed in their bulk counterparts. Accordingly, NP modified electrodes are of great significance for electrochemical applications [26–28]. In the light of the above ideas, an effective approach for the electrode modification is developed based on decorating graphene sheet with silver nanoparticles (AgNPs-G) and is used for the electrochemical determination of Aza. 2. Experimental 2.1. Materials and reagents All chemicals were of analytical reagent grade from Sigma Aldrich and used as received without further purification. Aza (N 99.0% purity), was kindly provided from kharazmi pharmaceutical company (Tehran, Iran). Tablets of MD-Azathioprine (50 mg per tablet) were purchased from Mehr Darou (Tehran, Iran). All aqueous solutions were prepared with DI water (Millipore Water Purification). Stock solutions of Aza were freshly prepared as required in 0.1 M of appropriate buffer solutions: 0.1 M acetate buffer solution for pHs 4 and 5, and 0.1 M phosphate buffer solution for pH values of 3, 6 and 7. 2.2. Apparatus A Philips CM30 transmission electron microscope (TEM) was used to study the morphology and structure of the sample. The X-ray diffraction (XRD) pattern was recorded using a STOE (STADI P) instrument operating with Cu–Kα radiation (λ = 1.54178 A°) at 40 kV/30 mA. A Thermo Nicolet Almega XR Raman spectrometer is applied to record the Raman spectra of the prepared samples. A Perkin-Elmer UV–vis spectrophotometer (Lambda 900) is used to investigate the absorption behavior of AgNPs-G nanosheets suspension. Voltammetric experiments are performed using a Potentiostat/Galvanostat (AUTOLAB 302). A conventional three-electrode system is used with a pyrolytic graphite working electrode (PGE, d = 3 mm, unmodified or modified), a saturated Ag/AgCl reference electrode and a Pt wire as the counter electrode.
3.1. Characterization of AgNPs-G As mentioned previously, the prepared graphene nanosheets decorated with Ag nanoparticles were characterized by means of transmission electron microscopy (TEM), X-ray diffraction (XRD), UV–vis and Raman spectroscopy techniques. As can be seen in the TEM images (Fig. 2), Ag nanoparticles with the average size of 5–50 nm are well deposited on the surface of graphene sheets. On the other hand, the characteristic absorption peak of silver nanoparticles was observed in the UV–vis spectrum of AgNPs (Fig. 3A). For graphene oxide two absorption peaks according to π → π⁎ and n → π⁎ transitions appeared at 235 and 306 nm, respectively. However, as the reduction reaction proceeded in the presence of Ag precursor, the π → π⁎ absorption peak displayed and a gradual red shift from 235 to 265 nm (associated with a decrease in intensity of n → π⁎ shoulder) and also a new peak at 400 nm is emerged, which is related to the AgNPs absorption. These observations revealed the reduction of GO nanosheets along with the deposition of AgNPs on its surface. XRD and Raman spectroscopy analysis are also performed for further characterization. The XRD pattern of AgNPs-G nanosheets that is shown in Fig. 3B, confirmed the immobilization of AgNPs on graphene nanosheets. The peaks that seen at 2θ of 38.11°, 44.33°, 64.5° and 77.46°, are corresponding to (111), (200), (220) and (311) crystal planes of metallic Ag, respectively. Fig. 4 indicates the Raman spectra of GO and AgNPs-G nanosheets. Apart from a slight shift in the characteristic D and G bands, an apparent increase in their intensities is observed for AgNPs-G, which is due to the surface enhancement effect (SERS) of metallic NPs, such as Ag or Au, on graphene sheets. Moreover, the ratio between D and G is clearly increased from ID/IG = 0.98 in the case of GO sheets to 1.2 for AgNPs-G. All these results together affirmed the deposition of AgNPs on the graphene surface. 3.2. Electrochemical investigation of Aza at Ag NPs-G/PGE As a pharmaceutically interesting electroactive compound, Azathioprine can be electrochemically reduced on the surface of the electrodes based on a four-electron reduction of its nitro functional group to the corresponding hydroxylamine (NHOH) described by the following equation [8]:
2.3. Preparation of the Ag NPs-G\PG modified electrode Graphene oxide (GO) nanosheets were synthesized by the modified Hummers method as reported in the literature [29]. For decorating graphene with silver nanoparticles, 50 mg GO and 0.9 mM AgNO3 in 200 mL DI water were mixed in a 500 mL four-necked flask under a nitrogen atmosphere and ultrasonicated for 1 h. Then, NaBH4 (50 mL, 13 mM) was added slowly as the reducing agent and the reaction mixture was left to stirred at 100 °C for 24 h. The solid product (AgNPs-G nanosheets) was isolated by repeated centrifugation at 13,000 rpm and washed by DI water several times. In a similar process, the reduced graphene was prepared under the same conditions only without Ag precursor. Prior to the electrode modification, the PGE was polished with 0.05 μm alumina slurry, rinsed thoroughly with DI water and then sonicated in ethanol and DI water each for 5 min, sequentially. The Ag
ð1Þ However, on the surface of bare electrodes such as glassy carbon or pyrolytic graphite only a very weak broad cathodic peak is observed at almost high overpotentials due to its sluggish electron transfer kinetics (Fig. 5a). Electrode surface modification with suitable electron transfer mediators results in improved catalytic activity and accordingly enhancement of the electrochemical response. As mentioned previously, herein the pyrolytic graphite electrode surface was modified with graphene nanosheets decorated with AgNPs (AgNPs-G) suspension. Taking advantage of high surface area provided by the graphene nanosheets as well as
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Fig. 2. TEM images of graphene sheet decorated with silver nanoparticles (AgNPs-G).
strong adsorption of Aza at AgNPs, the electrochemical response of Aza showed a remarkable enhancement on the surface of the modified electrode (AgNPs-G/PGE). As can be seen in Fig. 5b, by modifying the PGE surface with 20 μL of AgNPs-G suspension and after 50 s of accumulation time at open circuit condition, a well-defined reduction wave with a remarkable increase in its cathodic peak current from 20 to 500 μA along with a notable lowering of overpotential (175 mV) is observed. During the reverse scan no anodic peak was appeared for Aza, which suggests a totally irreversible behavior on the electrode surface. For comparison, we also modified the PGE surface with the same amount of graphene nanosheets under identical conditions (20 μL of G suspension and 50 s of accumulation time). As can be observed in Fig. 5c there is only a slight increase in the cathodic peak current (associated with higher capacitive current). One of the crucial aspects regarding to chemically reduced graphene oxide nanosheets is the restoration of π–π conjugated backbone which usually leads to restacking of graphene sheets. As a result, the dispersions aggregate in a time scale of days. Graphene nanosheets decorated with metal nanoparticles show better dispersibility due to the fact that the NPs can act as spacers between nanosheets and prevent further aggregation. The same results also observed for other carbon nanomaterials such as CNTs [30]. Herein, decoration of graphene nanosheets with Ag NPs giving a black dispersion that is quite stable for 3–4 months. The excellent dispersibility of the resulting product (AgNPs-G nanosheets) in water leads to the formation of a stable dispersion which is capable of forming
a uniform and stable thin film on the surface of the PGE. As a result, the diffusion regime of the electroactive species on the surface of the modified electrode changed and the background current decreased on the surface of the modified electrode. 3.3. Optimization of the amount of modifier and accumulation time The electrochemical response characteristics of the modified electrode (AgNPs-G/PGE) toward Aza's reduction can be directly affected by the amount of modifier used. In order to investigate the influence of the modifier, different amounts of AgNPs-G suspension (1 mg mL−1) was casted on the surface of PGE. The obtained cyclic voltammograms of Aza on the surface of the modified PGE revealed that by increasing the amount of the casted modifier suspension from 10 to 15 and consequently to 20 μL the cathodic peak current increased quickly, which can be related to the enlargement of the electrode microscopic surface area. However for further amounts of the modifier (25 μL) no obvious increase in Aza's reduction peak observed due to the undesired mechanical properties of the modifier layer and also the sluggish electron transfer kinetics on the surface of the electrode modified with a thick layer of modifier. As a result, 20 μL of 1 mg mL−1 AgNPs-G suspension is choose as the optimum amount of the modifier used for the preparation of the modified electrode. In order to study the effect of Ag NPs, we investigated the effect of accumulation time on the surface of PGE modified with G and AgNPs-G
Fig. 3. (A) UV–vis spectrum of aqueous solution of graphene oxide (GO) and graphene sheets decorated with AgNPs and (B) XRD pattern of graphene sheets decorated with AgNPs.
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an accumulation step at open circuit condition even at a very short period of 10 s has a great influence on the electrochemical reduction of Aza. It can be seen that by increasing the accumulation time from 10 to 50 s the cathodic peak current increased owing to the fact that a longer accumulation time caused that higher amounts of Aza adsorbed on the surface of the electrode. However further prolonging of the accumulation time from 50 to 70 s is only associated with a slight increase in peak height. Moreover at 100 s the peak current was levelled off, which is attributed to the surface saturation of the modified electrode. As a result, an accumulation time of 50 s was set as the optimum condition and used in the following experiments. 3.4. Influence of pH on the voltammetric responses
Fig. 4. Raman spectra of graphene sheets (black line) and graphene sheets decorated with AgNPs (red line).
nanosheets (Fig. 6A). There is a slight increase on the surface of PGE modified with graphene nanosheets by applying 50 s accumulation time which might be due to the π–π interaction of Aza and graphene backbone. However, the major increase in the cathodic peak current of Aza was observed on the surface of electrode modified with graphene nanosheets decorated with Ag nanoparticles along with a remarkable decrease in Aza's overpotential which can be attributed to the presence of Ag nanoparticles on the surface of graphene nanosheets and the adsorption of Aza molecules on the surface of the deposited nanoparticles. To study this assumption, the effect of accumulation time on the response characteristics of Aza on the surface of AgNPs-G/PGE was investigated. Based on the fact that thiolic compounds can be strongly adsorbed on the surface of Au and Ag substrates, it is assumed that applying a certain accumulation period can remarkably enhance the electrode response characteristics. As clearly shown in Fig. 6B, exerting
Since the redox reaction of Aza is associated with the proton (H+ ions) transfer as shown in Eq. (1), the pH of the supporting electrolyte has a significance influence on its electrochemical behavior. To investigate the effect of the pH on the electro-reduction of Aza at the modified electrode, cyclic voltammograms of 0.1 mM Aza at different pH values in the range of 3.0 to 7.0 are recorded at a potential scan rate of 100 mV s−1 (Fig. 7A). It was found that with increasing the pH value of the supporting electrolyte, the peak potential shifted negatively suggesting the participation of H+ ions in the reduction reaction. The plot of Ep,c versus pH values shows a good linear relationship (with R2 = 0.9984) described by the following equation: Ep;c ðmVÞ ¼ −275:9−55:65 pH
ð2Þ
The slope of the linear variation of Ep,c versus pH showed a value of about − 55.65 mV per pH unit, which clearly indicates that equal numbers of electrons and protons are involved in the reduction of Aza on the surface of the AgNPs-G/PG modified electrode. On the other hand, by raising pH values from 3.0 to 7.0 the cathodic peak current gradually increased and reaching to its maximum at pH = 6.0. Further increase of the pH to 7.0 caused the peak height to decrease (Fig. 7B). Hence, phosphate buffer solution of pH 6.0 was choose as the supporting electrolyte and used for all voltammetric studies. 3.5. Effect of potential scan rate In order to study the electrochemical mechanism of the redox reactions including diffusion controlled, surface processed and adsorptiondiffusion controlled process, cyclic voltammograms are recorded on the surface of the modified electrode at different potential scan rates. The nature of the electrode process on the surface of AgNPs-G/PGE is investigated for 0.1 mM Aza at pH 6.0 in various scan rates (in the range of 25 to 350 mV s−1) and is shown in Fig. 8A. As can be seen, the reduction peak current shifts negatively with increasing the scan rate. The plot of the logarithm of the peak current versus logarithm of the scan rate shows a linear relationship with the regression equation described as follows: log ip;c ¼ 0:7293 logðυÞ þ 0:5079 R2 ¼ 0:9986; ip;c : μA; υ : mVs−1 : ð3Þ A slope of 0.7293 for the linear variation of the log (ip,c) and log(υ) (Fig. 7B) indicates a mixed adsorption/diffusion-controlled process on the surface of the modified electrode [30]. On the other hand, as can be seen in Fig. 8A, only a reduction peak is observed even at low scan rates, which suggests a totally irreversible behavior of Aza on the surface of the electrode. These observations were also confirmed by the linear relationship obtained between Ep,c and the logarithm of the scan rate as in Eq. (4):
Fig. 5. Cyclic voltammograms of bare PGE (a), AgNPs-G/PGE (b) and graphene coated PGE (c) in 0.1 M PBS (pH 6.0) containing 0.1 mM Aza, scan rate was 100 mV s−1.
Ep;c ¼ −219:38 logυ–149:73
R2 ¼ 0:9949; Ep;c : mV; υ : mVs−1 : ð4Þ
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Fig. 6. (A) The effect of presence of Ag nanoparticles on the surface of graphene nanosheets at bare PGE, graphene coated PGE and AgNPs-G/PGE (after 0 and 50 s of accumulation time) and (B) cyclic voltammograms on the surface of AgNPs-G/PGE at different accumulation times from 0 to 100 s (the inset: plot of ip,c versus taccumulation) for 0.1 mM Aza, the scan rate was 100 mV s−1.
3.6. Voltammetric determinations The calibration curve for Aza determination was established by recording the cathodic peak currents of Aza at different concentrations using AgNPs-G/PG modified electrode (Fig. 9A). Under the previously mentioned optimum conditions (0.1 M phosphate buffer solution of pH 6.0 and an accumulation time of 50 s), the reduction peak currents were proportional to Aza concentrations over 3 intervals in the range of 7 × 10−7 to 1 × 10−4 mol L−1 (Eq. (5)): ip;c ðμAÞ ¼ 4:7434C Aza ðμMÞ þ 33:527
R2 ¼ 0:9988
ð5Þ
which is one order of magnitude larger than previous reports on electrochemical determination of Aza [7, 8]. Based on the linear plot of peak current versus Aza's concentration (Fig. 9B), a detection limit (S/N = 3) of 68 nM is obtained for Aza determination, indicating the system is quite sensitive toward Aza.
Table 1 compares some performance characteristics of the prepared modified electrode with those of previous works reported for the electrochemical determination of Aza. As can be seen, the proposed method provides a wider linear dynamic range and a lower detection limit in most cases. Moreover, as previously mentioned, other analytical techniques such as chromatographic and spectrometric methods require expensive instrumentations and tedious sample preparations (e.g. extraction, filtration and preconcentration) that make them inappropriate for regular analysis. The proposed sensing platform is not only simple to prepare, but also cost effective and time efficient, which are all beneficial for routine assays. 3.7. Determination of Aza in pharmaceutical preparations To assess the applicability of the proposed method in clinical preparations, the prepared modified electrode was used for the determination of the content of Aza in Azathioprine tablets as real pharmaceutical samples.
Fig. 7. (A) Cyclic voltammograms of 0.1 mM Aza at the AgNPs-G/PGE in various pHs (from 3 to 7) of buffer solution (Inset: dependence of Ep,c on pH solution) and (B) plot of Ip,c versus pH values of the electrolyte, the potential scan rate was 100 mV s−1.
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Fig. 8. (A) Cyclic voltammograms of 0.1 mM Aza at the AgNPs-G/PGE at different scan rates in 0.1 M PBS (pH 6.0); (B) the plot of log(ip,c) versus log(υ) and (C) variation of peak potential (Ep,c) with log(υ).
Fig. 9. (A) CVs for various concentrations of Aza in the range of (up to down) 0.7–100 μmol L−1 in 0.1 M phosphate buffer solution (pH 6.0) and (B) corresponding linear calibration curve of peak current vs. Aza concentration; scan rate was 100 mV s−1.
Three tablets containing labeled amounts of 50 mg Aza were accurately weighted and ground to a fine powder. From this powder, different concentrations of Aza were prepared and consequently the cyclic voltammograms of them on the surface of AgNPs-G/PGE were investigated (Fig. 10A). As clearly shown in the Fig. 10B, the calibration curve of ip,c vs. CAza in tablet samples show a good linear relationship with the regression coefficient of R2 = 0.9981 with the same trend obtained for Aza,
Table 1 Comparison of some analytical characteristics of the prepared sensor with those of other electrochemical methods reported for the determination of Aza. Method\electrode
Linear range (μM)
LOD (μM)
Reference
CV \ Nanodiamond–graphite-chitosan modified GCE CV \ Carbon nanoparticles modified GCE CV \ Gold NPs modified Au electrode CV \ Graphene-chitosan modified GCE
0.2–1 and 1–100
0.065
[7]
0.2–2 and 1–50 0.095–900 0.1–1.96 and 1.96–26.7 0.1–100
0.080 0.090 0.470
[8] [31] [32]
0.068
This work
CV \ Ag NPs@Graphene modified PGE
which indicates that the proposed sensing platform can be successfully applied for sensitive detection of Aza in practical applications. 4. Conclusions Graphene nanosheets were used as a supporting substrate for deposition of Ag nanoparticles. Using this suspension, a modified pyrolytic graphite electrode was used for electrochemical determination of Aza. It has been demonstrated that the prepared modified electrode showed an efficient catalytic role toward the electroreduction of Aza, which led to a remarkable increase in the cathodic peak current (~ 25 times) as well as noticeable decrease in its reduction overpotential (~175 mV). These observations are related to the enlargement of specific surface area of the electrode owing to the presence of graphene sheets along with effective accumulation of Aza on the surface of Ag NPs. The prepared modified electrode is successfully applied for the voltammetric determination of Aza in pharmaceutical samples with a good linear relationship between the cathodic peak current (Ip,c) and the concentration of Aza in the range of 7 × 10−7 to 1 × 10−4 mol L− 1. High sensitivity and low detection limit obtained by this sensing platform
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Fig. 10. (A) Cyclic voltammograms for solutions containing different amount of Aza tablet in the range of (up to down) 10–100 μM in 0.1 M PBS (pH 6.0) and (B) corresponding linear calibration curves of peak current versus Aza concentration; scan rate was 100 mV s−1.
make it a promising candidate for the determination of Aza in pharmaceutical formulations and clinical preparations. Acknowledgements The authors gratefully acknowledge the support of this work by the Research Council and the Center of Excellence for Nanostructures of the Sharif University of Technology, Tehran, Iran.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Voltammetric studies of Azathioprine on the surface of graphite electrode modified with graphene nanosheets decorated with Ag nanoparticles Elham Asadian a, Azam Iraji zad a,b, Saeed Shahrokhian a,c,⁎ a b c
Institute for Nanoscience and Nanotechnology (INST), Sharif University of Technology, Tehran, Iran Department of Physics, Sharif University of Technology, Tehran 14588-89694, Iran Department of Chemistry, Sharif University of Technology, Tehran 11155-9516, Iran
a r t i c l e
i n f o
Article history: Received 22 May 2015 Received in revised form 31 July 2015 Accepted 7 September 2015 Available online 12 September 2015 Keywords: Graphene nanosheets Ag Nanoparticles Modified electrode Azathioprine Voltammetry
a b s t r a c t By using graphene nanosheets decorated with Ag nanoparticles (AgNPs-G) as an effective approach for the surface modification of pyrolytic graphite electrode (PGE), a sensing platform was fabricated for the sensitive voltammetric determination of Azathioprine (Aza). The prepared AgNPs-G nanosheets were characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), UV–vis and Raman spectroscopy techniques. The electrochemical behavior of Aza was investigated by means of cyclic voltammetry. Comparing to the bare PGE, a remarkable enhancement was observed in the response characteristics of Aza on the surface of the modified electrode (AgNPs-G/PGE) as well as a noticeable decrease in its reduction overpotential. These results can be attributed to the incredible enlargement in the microscopic surface area of the electrode due to the presence of graphene nanosheets together with strong adsorption of Aza on its surface. The effect of experimental parameters such as accumulation time, the amount of modifier suspension and pH of the supporting electrolyte were also optimized toward obtaining the maximum sensitivity. Under the optimum conditions, the calibration curve studies demonstrated that the peak current increased linearly with Aza concentrations in the range of 7 × 10−7 to 1 × 10−4 mol L−1 with the detection limit of 68 nM. Further experiments revealed that the modified electrode can be successfully applied for the accurate determination of Aza in pharmaceutical preparations. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Azathioprine (Aza) (Fig. 1), belongs to the chemical class of purine analogues, is an immunosuppressive drug used in organ transplantation (usually kidney or liver) along with other medications to prevent new organ transplant immunological rejection. It is also used to treat an array of autoimmune diseases including rheumatoid arthritis [1]. Aza is a prodrug (a precursor of a drug), which is converted in the body to its active form called mercaptopurine and acts by decreasing the effects of certain cells in the body's immune system [2]. However, long term use of Aza increases the risk of developing certain types of cancer such as leukemia, lymphoma and skin cancer. Hence careful and routine monitoring of Aza's concentration is of great importance in clinical practices. A brief literature review reveals that various analytical techniques such as high performance liquid chromatography (HPLC) [3, 4], 1HNMR spectroscopy [5] and spectrophotometry [6] have been used for the determination of Aza in pharmaceutical formulations and biological fluids. Although these methods provide accurate measurements, they ⁎ Corresponding author at: Institute for Nanoscience and Nanotechnology (INST), Sharif University of Technology, Tehran, Iran. E-mail address: [email protected] (S. Shahrokhian).
http://dx.doi.org/10.1016/j.msec.2015.09.022 0928-4931/© 2015 Elsevier B.V. All rights reserved.
suffer from some disadvantages such as expensive instrumentations, tedious sample preparations, time consuming and low sensitivity and/or selectivity that make them inappropriate for regular analysis. Regarding to these issues a precise and accurate method for routine assays is of great importance. Electrochemical techniques come up with numerous advantages including low cost, simplicity, short analysis time and sensitivity. However electro-reduction of Aza at the surface of bare glassy carbon or pyrolytic graphite electrodes only shows a very weak and broad peak, revealing high overpotential values in its electrode process [7, 8]. One way to address this issue and improve the slow electron transfer kinetics as well as increase the active electrode surface area is to modify the electrode surface with various modifiers. From this point of view, nanomaterials such as nanotubes [9, 10], nanoparticles [11, 12], nanosheets [13–15] and so on are promising candidates. Graphene, a 2D one atom thick carbon nanosheet, have attracted a great deal of attention since its discovery in 2004 due to its outstanding properties: high electrical conductivity, large specific surface area (theoretical value ≈ 2630 m2 g−1), thermal and chemical stability and extraordinary mechanical strength [16]. All these properties together along with its 2D structure make graphene pioneer in various applications including energy storage devices (supercapacitors [17], batteries [18]), solar cells [19] and sensors [20–22]. Moreover, decorating graphene with other nanomaterials such as nanorods or nanoparticles provides
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NPs-G/modified PGE was prepared by casting a desired volume (20 μL in optimum condition) of the above mentioned AgNPs-G suspension on the PGE surface using a micropipette and left to dry at room temperature. Before the voltammetric measurements, the modified electrode was cycled between 0 and −1 V (scan rate 100 mV s−1) in 0.1 M phosphate buffer solution (pH 6.0) for several times until acquiring a stable background. 3. Results and discussion Fig. 1. Molecular structure of Azathioprine.
prominent properties, due to the synergetic effect between them [23–25]. Nanoparticles (NPs), usually exhibit size-related properties that differ significantly from those observed in their bulk counterparts. Accordingly, NP modified electrodes are of great significance for electrochemical applications [26–28]. In the light of the above ideas, an effective approach for the electrode modification is developed based on decorating graphene sheet with silver nanoparticles (AgNPs-G) and is used for the electrochemical determination of Aza. 2. Experimental 2.1. Materials and reagents All chemicals were of analytical reagent grade from Sigma Aldrich and used as received without further purification. Aza (N 99.0% purity), was kindly provided from kharazmi pharmaceutical company (Tehran, Iran). Tablets of MD-Azathioprine (50 mg per tablet) were purchased from Mehr Darou (Tehran, Iran). All aqueous solutions were prepared with DI water (Millipore Water Purification). Stock solutions of Aza were freshly prepared as required in 0.1 M of appropriate buffer solutions: 0.1 M acetate buffer solution for pHs 4 and 5, and 0.1 M phosphate buffer solution for pH values of 3, 6 and 7. 2.2. Apparatus A Philips CM30 transmission electron microscope (TEM) was used to study the morphology and structure of the sample. The X-ray diffraction (XRD) pattern was recorded using a STOE (STADI P) instrument operating with Cu–Kα radiation (λ = 1.54178 A°) at 40 kV/30 mA. A Thermo Nicolet Almega XR Raman spectrometer is applied to record the Raman spectra of the prepared samples. A Perkin-Elmer UV–vis spectrophotometer (Lambda 900) is used to investigate the absorption behavior of AgNPs-G nanosheets suspension. Voltammetric experiments are performed using a Potentiostat/Galvanostat (AUTOLAB 302). A conventional three-electrode system is used with a pyrolytic graphite working electrode (PGE, d = 3 mm, unmodified or modified), a saturated Ag/AgCl reference electrode and a Pt wire as the counter electrode.
3.1. Characterization of AgNPs-G As mentioned previously, the prepared graphene nanosheets decorated with Ag nanoparticles were characterized by means of transmission electron microscopy (TEM), X-ray diffraction (XRD), UV–vis and Raman spectroscopy techniques. As can be seen in the TEM images (Fig. 2), Ag nanoparticles with the average size of 5–50 nm are well deposited on the surface of graphene sheets. On the other hand, the characteristic absorption peak of silver nanoparticles was observed in the UV–vis spectrum of AgNPs (Fig. 3A). For graphene oxide two absorption peaks according to π → π⁎ and n → π⁎ transitions appeared at 235 and 306 nm, respectively. However, as the reduction reaction proceeded in the presence of Ag precursor, the π → π⁎ absorption peak displayed and a gradual red shift from 235 to 265 nm (associated with a decrease in intensity of n → π⁎ shoulder) and also a new peak at 400 nm is emerged, which is related to the AgNPs absorption. These observations revealed the reduction of GO nanosheets along with the deposition of AgNPs on its surface. XRD and Raman spectroscopy analysis are also performed for further characterization. The XRD pattern of AgNPs-G nanosheets that is shown in Fig. 3B, confirmed the immobilization of AgNPs on graphene nanosheets. The peaks that seen at 2θ of 38.11°, 44.33°, 64.5° and 77.46°, are corresponding to (111), (200), (220) and (311) crystal planes of metallic Ag, respectively. Fig. 4 indicates the Raman spectra of GO and AgNPs-G nanosheets. Apart from a slight shift in the characteristic D and G bands, an apparent increase in their intensities is observed for AgNPs-G, which is due to the surface enhancement effect (SERS) of metallic NPs, such as Ag or Au, on graphene sheets. Moreover, the ratio between D and G is clearly increased from ID/IG = 0.98 in the case of GO sheets to 1.2 for AgNPs-G. All these results together affirmed the deposition of AgNPs on the graphene surface. 3.2. Electrochemical investigation of Aza at Ag NPs-G/PGE As a pharmaceutically interesting electroactive compound, Azathioprine can be electrochemically reduced on the surface of the electrodes based on a four-electron reduction of its nitro functional group to the corresponding hydroxylamine (NHOH) described by the following equation [8]:
2.3. Preparation of the Ag NPs-G\PG modified electrode Graphene oxide (GO) nanosheets were synthesized by the modified Hummers method as reported in the literature [29]. For decorating graphene with silver nanoparticles, 50 mg GO and 0.9 mM AgNO3 in 200 mL DI water were mixed in a 500 mL four-necked flask under a nitrogen atmosphere and ultrasonicated for 1 h. Then, NaBH4 (50 mL, 13 mM) was added slowly as the reducing agent and the reaction mixture was left to stirred at 100 °C for 24 h. The solid product (AgNPs-G nanosheets) was isolated by repeated centrifugation at 13,000 rpm and washed by DI water several times. In a similar process, the reduced graphene was prepared under the same conditions only without Ag precursor. Prior to the electrode modification, the PGE was polished with 0.05 μm alumina slurry, rinsed thoroughly with DI water and then sonicated in ethanol and DI water each for 5 min, sequentially. The Ag
ð1Þ However, on the surface of bare electrodes such as glassy carbon or pyrolytic graphite only a very weak broad cathodic peak is observed at almost high overpotentials due to its sluggish electron transfer kinetics (Fig. 5a). Electrode surface modification with suitable electron transfer mediators results in improved catalytic activity and accordingly enhancement of the electrochemical response. As mentioned previously, herein the pyrolytic graphite electrode surface was modified with graphene nanosheets decorated with AgNPs (AgNPs-G) suspension. Taking advantage of high surface area provided by the graphene nanosheets as well as
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Fig. 2. TEM images of graphene sheet decorated with silver nanoparticles (AgNPs-G).
strong adsorption of Aza at AgNPs, the electrochemical response of Aza showed a remarkable enhancement on the surface of the modified electrode (AgNPs-G/PGE). As can be seen in Fig. 5b, by modifying the PGE surface with 20 μL of AgNPs-G suspension and after 50 s of accumulation time at open circuit condition, a well-defined reduction wave with a remarkable increase in its cathodic peak current from 20 to 500 μA along with a notable lowering of overpotential (175 mV) is observed. During the reverse scan no anodic peak was appeared for Aza, which suggests a totally irreversible behavior on the electrode surface. For comparison, we also modified the PGE surface with the same amount of graphene nanosheets under identical conditions (20 μL of G suspension and 50 s of accumulation time). As can be observed in Fig. 5c there is only a slight increase in the cathodic peak current (associated with higher capacitive current). One of the crucial aspects regarding to chemically reduced graphene oxide nanosheets is the restoration of π–π conjugated backbone which usually leads to restacking of graphene sheets. As a result, the dispersions aggregate in a time scale of days. Graphene nanosheets decorated with metal nanoparticles show better dispersibility due to the fact that the NPs can act as spacers between nanosheets and prevent further aggregation. The same results also observed for other carbon nanomaterials such as CNTs [30]. Herein, decoration of graphene nanosheets with Ag NPs giving a black dispersion that is quite stable for 3–4 months. The excellent dispersibility of the resulting product (AgNPs-G nanosheets) in water leads to the formation of a stable dispersion which is capable of forming
a uniform and stable thin film on the surface of the PGE. As a result, the diffusion regime of the electroactive species on the surface of the modified electrode changed and the background current decreased on the surface of the modified electrode. 3.3. Optimization of the amount of modifier and accumulation time The electrochemical response characteristics of the modified electrode (AgNPs-G/PGE) toward Aza's reduction can be directly affected by the amount of modifier used. In order to investigate the influence of the modifier, different amounts of AgNPs-G suspension (1 mg mL−1) was casted on the surface of PGE. The obtained cyclic voltammograms of Aza on the surface of the modified PGE revealed that by increasing the amount of the casted modifier suspension from 10 to 15 and consequently to 20 μL the cathodic peak current increased quickly, which can be related to the enlargement of the electrode microscopic surface area. However for further amounts of the modifier (25 μL) no obvious increase in Aza's reduction peak observed due to the undesired mechanical properties of the modifier layer and also the sluggish electron transfer kinetics on the surface of the electrode modified with a thick layer of modifier. As a result, 20 μL of 1 mg mL−1 AgNPs-G suspension is choose as the optimum amount of the modifier used for the preparation of the modified electrode. In order to study the effect of Ag NPs, we investigated the effect of accumulation time on the surface of PGE modified with G and AgNPs-G
Fig. 3. (A) UV–vis spectrum of aqueous solution of graphene oxide (GO) and graphene sheets decorated with AgNPs and (B) XRD pattern of graphene sheets decorated with AgNPs.
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an accumulation step at open circuit condition even at a very short period of 10 s has a great influence on the electrochemical reduction of Aza. It can be seen that by increasing the accumulation time from 10 to 50 s the cathodic peak current increased owing to the fact that a longer accumulation time caused that higher amounts of Aza adsorbed on the surface of the electrode. However further prolonging of the accumulation time from 50 to 70 s is only associated with a slight increase in peak height. Moreover at 100 s the peak current was levelled off, which is attributed to the surface saturation of the modified electrode. As a result, an accumulation time of 50 s was set as the optimum condition and used in the following experiments. 3.4. Influence of pH on the voltammetric responses
Fig. 4. Raman spectra of graphene sheets (black line) and graphene sheets decorated with AgNPs (red line).
nanosheets (Fig. 6A). There is a slight increase on the surface of PGE modified with graphene nanosheets by applying 50 s accumulation time which might be due to the π–π interaction of Aza and graphene backbone. However, the major increase in the cathodic peak current of Aza was observed on the surface of electrode modified with graphene nanosheets decorated with Ag nanoparticles along with a remarkable decrease in Aza's overpotential which can be attributed to the presence of Ag nanoparticles on the surface of graphene nanosheets and the adsorption of Aza molecules on the surface of the deposited nanoparticles. To study this assumption, the effect of accumulation time on the response characteristics of Aza on the surface of AgNPs-G/PGE was investigated. Based on the fact that thiolic compounds can be strongly adsorbed on the surface of Au and Ag substrates, it is assumed that applying a certain accumulation period can remarkably enhance the electrode response characteristics. As clearly shown in Fig. 6B, exerting
Since the redox reaction of Aza is associated with the proton (H+ ions) transfer as shown in Eq. (1), the pH of the supporting electrolyte has a significance influence on its electrochemical behavior. To investigate the effect of the pH on the electro-reduction of Aza at the modified electrode, cyclic voltammograms of 0.1 mM Aza at different pH values in the range of 3.0 to 7.0 are recorded at a potential scan rate of 100 mV s−1 (Fig. 7A). It was found that with increasing the pH value of the supporting electrolyte, the peak potential shifted negatively suggesting the participation of H+ ions in the reduction reaction. The plot of Ep,c versus pH values shows a good linear relationship (with R2 = 0.9984) described by the following equation: Ep;c ðmVÞ ¼ −275:9−55:65 pH
ð2Þ
The slope of the linear variation of Ep,c versus pH showed a value of about − 55.65 mV per pH unit, which clearly indicates that equal numbers of electrons and protons are involved in the reduction of Aza on the surface of the AgNPs-G/PG modified electrode. On the other hand, by raising pH values from 3.0 to 7.0 the cathodic peak current gradually increased and reaching to its maximum at pH = 6.0. Further increase of the pH to 7.0 caused the peak height to decrease (Fig. 7B). Hence, phosphate buffer solution of pH 6.0 was choose as the supporting electrolyte and used for all voltammetric studies. 3.5. Effect of potential scan rate In order to study the electrochemical mechanism of the redox reactions including diffusion controlled, surface processed and adsorptiondiffusion controlled process, cyclic voltammograms are recorded on the surface of the modified electrode at different potential scan rates. The nature of the electrode process on the surface of AgNPs-G/PGE is investigated for 0.1 mM Aza at pH 6.0 in various scan rates (in the range of 25 to 350 mV s−1) and is shown in Fig. 8A. As can be seen, the reduction peak current shifts negatively with increasing the scan rate. The plot of the logarithm of the peak current versus logarithm of the scan rate shows a linear relationship with the regression equation described as follows: log ip;c ¼ 0:7293 logðυÞ þ 0:5079 R2 ¼ 0:9986; ip;c : μA; υ : mVs−1 : ð3Þ A slope of 0.7293 for the linear variation of the log (ip,c) and log(υ) (Fig. 7B) indicates a mixed adsorption/diffusion-controlled process on the surface of the modified electrode [30]. On the other hand, as can be seen in Fig. 8A, only a reduction peak is observed even at low scan rates, which suggests a totally irreversible behavior of Aza on the surface of the electrode. These observations were also confirmed by the linear relationship obtained between Ep,c and the logarithm of the scan rate as in Eq. (4):
Fig. 5. Cyclic voltammograms of bare PGE (a), AgNPs-G/PGE (b) and graphene coated PGE (c) in 0.1 M PBS (pH 6.0) containing 0.1 mM Aza, scan rate was 100 mV s−1.
Ep;c ¼ −219:38 logυ–149:73
R2 ¼ 0:9949; Ep;c : mV; υ : mVs−1 : ð4Þ
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Fig. 6. (A) The effect of presence of Ag nanoparticles on the surface of graphene nanosheets at bare PGE, graphene coated PGE and AgNPs-G/PGE (after 0 and 50 s of accumulation time) and (B) cyclic voltammograms on the surface of AgNPs-G/PGE at different accumulation times from 0 to 100 s (the inset: plot of ip,c versus taccumulation) for 0.1 mM Aza, the scan rate was 100 mV s−1.
3.6. Voltammetric determinations The calibration curve for Aza determination was established by recording the cathodic peak currents of Aza at different concentrations using AgNPs-G/PG modified electrode (Fig. 9A). Under the previously mentioned optimum conditions (0.1 M phosphate buffer solution of pH 6.0 and an accumulation time of 50 s), the reduction peak currents were proportional to Aza concentrations over 3 intervals in the range of 7 × 10−7 to 1 × 10−4 mol L−1 (Eq. (5)): ip;c ðμAÞ ¼ 4:7434C Aza ðμMÞ þ 33:527
R2 ¼ 0:9988
ð5Þ
which is one order of magnitude larger than previous reports on electrochemical determination of Aza [7, 8]. Based on the linear plot of peak current versus Aza's concentration (Fig. 9B), a detection limit (S/N = 3) of 68 nM is obtained for Aza determination, indicating the system is quite sensitive toward Aza.
Table 1 compares some performance characteristics of the prepared modified electrode with those of previous works reported for the electrochemical determination of Aza. As can be seen, the proposed method provides a wider linear dynamic range and a lower detection limit in most cases. Moreover, as previously mentioned, other analytical techniques such as chromatographic and spectrometric methods require expensive instrumentations and tedious sample preparations (e.g. extraction, filtration and preconcentration) that make them inappropriate for regular analysis. The proposed sensing platform is not only simple to prepare, but also cost effective and time efficient, which are all beneficial for routine assays. 3.7. Determination of Aza in pharmaceutical preparations To assess the applicability of the proposed method in clinical preparations, the prepared modified electrode was used for the determination of the content of Aza in Azathioprine tablets as real pharmaceutical samples.
Fig. 7. (A) Cyclic voltammograms of 0.1 mM Aza at the AgNPs-G/PGE in various pHs (from 3 to 7) of buffer solution (Inset: dependence of Ep,c on pH solution) and (B) plot of Ip,c versus pH values of the electrolyte, the potential scan rate was 100 mV s−1.
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Fig. 8. (A) Cyclic voltammograms of 0.1 mM Aza at the AgNPs-G/PGE at different scan rates in 0.1 M PBS (pH 6.0); (B) the plot of log(ip,c) versus log(υ) and (C) variation of peak potential (Ep,c) with log(υ).
Fig. 9. (A) CVs for various concentrations of Aza in the range of (up to down) 0.7–100 μmol L−1 in 0.1 M phosphate buffer solution (pH 6.0) and (B) corresponding linear calibration curve of peak current vs. Aza concentration; scan rate was 100 mV s−1.
Three tablets containing labeled amounts of 50 mg Aza were accurately weighted and ground to a fine powder. From this powder, different concentrations of Aza were prepared and consequently the cyclic voltammograms of them on the surface of AgNPs-G/PGE were investigated (Fig. 10A). As clearly shown in the Fig. 10B, the calibration curve of ip,c vs. CAza in tablet samples show a good linear relationship with the regression coefficient of R2 = 0.9981 with the same trend obtained for Aza,
Table 1 Comparison of some analytical characteristics of the prepared sensor with those of other electrochemical methods reported for the determination of Aza. Method\electrode
Linear range (μM)
LOD (μM)
Reference
CV \ Nanodiamond–graphite-chitosan modified GCE CV \ Carbon nanoparticles modified GCE CV \ Gold NPs modified Au electrode CV \ Graphene-chitosan modified GCE
0.2–1 and 1–100
0.065
[7]
0.2–2 and 1–50 0.095–900 0.1–1.96 and 1.96–26.7 0.1–100
0.080 0.090 0.470
[8] [31] [32]
0.068
This work
CV \ Ag NPs@Graphene modified PGE
which indicates that the proposed sensing platform can be successfully applied for sensitive detection of Aza in practical applications. 4. Conclusions Graphene nanosheets were used as a supporting substrate for deposition of Ag nanoparticles. Using this suspension, a modified pyrolytic graphite electrode was used for electrochemical determination of Aza. It has been demonstrated that the prepared modified electrode showed an efficient catalytic role toward the electroreduction of Aza, which led to a remarkable increase in the cathodic peak current (~ 25 times) as well as noticeable decrease in its reduction overpotential (~175 mV). These observations are related to the enlargement of specific surface area of the electrode owing to the presence of graphene sheets along with effective accumulation of Aza on the surface of Ag NPs. The prepared modified electrode is successfully applied for the voltammetric determination of Aza in pharmaceutical samples with a good linear relationship between the cathodic peak current (Ip,c) and the concentration of Aza in the range of 7 × 10−7 to 1 × 10−4 mol L− 1. High sensitivity and low detection limit obtained by this sensing platform
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Fig. 10. (A) Cyclic voltammograms for solutions containing different amount of Aza tablet in the range of (up to down) 10–100 μM in 0.1 M PBS (pH 6.0) and (B) corresponding linear calibration curves of peak current versus Aza concentration; scan rate was 100 mV s−1.
make it a promising candidate for the determination of Aza in pharmaceutical formulations and clinical preparations. Acknowledgements The authors gratefully acknowledge the support of this work by the Research Council and the Center of Excellence for Nanostructures of the Sharif University of Technology, Tehran, Iran.
[13] [14] [15] [16] [17] [18] [19] [20] [21]
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