Green electrodeposition of gold hierarchical dendrites of pyramidal nanoparticles and determination of azathioprine

Sensors and Actuators B 215 (2015) 113–118

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Green electrodeposition of gold hierarchical dendrites of pyramidal nanoparticles and determination of azathioprine R. Dehdari Vais a , N. Sattarahmady a,b,c,∗ , K. Karimian d , H. Heli a,c,∗ a

Nanomedicine and Nanobiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran Department of Medical Physics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran Department of Nanomedicine, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran d Andisheh Pharma Sciences R&D Inc., Yousefabad, Jahanarar Avenue, Tehran, Iran b c

a r t i c l e

i n f o

Article history: Received 22 November 2014 Received in revised form 28 February 2015 Accepted 4 March 2015 Available online 1 April 2015 Keywords: Gold Hierarchical nanostructure Electrodeposition Green synthesis Azathioprine Electroanalysis

a b s t r a c t Gold hierarchical dendrites consisting of an array of parallel-arranged pyramidal nanoparticles were electrodeposited in the presence of lysine on a gold surface and characterized by scanning electron microscopy. The kinetics of azathioprine electroreduction on the dendrites’ surface was studied in a phosphate buffer solution, pH 7.40 by voltammetric measurements. Azathioprine was electroreduced on the dendrites’ surface at a lower potential and with a higher rate, compared to a polycrystalline smooth gold surface through an irreversible process. Based on the results, a sensitive electrochemical sensor was designed based on the gold hierarchical dendrites for the determination of azathioprine. A differential pulse voltammetric procedure was developed for determination of azathioprine. A linear dynamic range of 0.095–900 ␮mol L−1 with a calibration sensitivity of 0.14 A L mol−1 cm−2 , and a detection limit of 0.090 ␮mol L−1 was obtained. The differential pulse voltammetry method was also applied to the analysis of azathioprine tablets. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Azathioprine (6-[(1-methyl-4-nitro-1H-imidazol-5-yl)sulfanyl] -7H-purine, Scheme 1) is an immunosuppressant and antileukemic drug. It is used in allotransplantation procedures, systemic anti-inflammatory states (e.g., lupus erythematosus, rheumatoid arthritis, Crohn’s disease and polymyosites) and prevention of rejection following organ transplantation through inhibition the T and B cell proliferations [1–3]. Drug analysis methods have had extensive impacts on public health and play an important role in the stability, toxicology, formulation, bioequivalency and phamakokinetics and pharmacodynamics studies of drugs [4–8]. Study of the redox properties of drugs and the drug’s electrode mechanisms can provide insight into their metabolic fate and their in vivo pharmacological activity [9,10].

∗ Corresponding authors at: Nanomedicine and Nanobiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. Tel.: +98 71 32 34 93 32; fax: +98 71 32 34 93 32. E-mail addresses: [email protected], [email protected] (N. Sattarahmady), [email protected], [email protected] (H. Heli). http://dx.doi.org/10.1016/j.snb.2015.03.014 0925-4005/© 2015 Elsevier B.V. All rights reserved.

A number of techniques have been proposed for the determination of azathioprine. To this end, spectrophotometry [11], chemiluminescence [12], high-performance liquid chromatography [13,14], 1 H NMR spectroscopy [15], high-performance thin-layer chromatography [16], ultra performance liquid chromatography [17], capillary zone electrophoresis [18], and surfaceenhanced Raman spectroscopy [19] have been reported for the determination of azathioprine. However, some of these methods suffer from various disadvantages, such as sensitivity and selectivity, high costs, long analysis times, or cumbersome extraction procedures. Nanostructured materials have attracted much attention due to the unique size- and shape-dependent properties related to their small dimensions and quantum size effects [20]. New nanostructured materials represent the novel properties of electronic conductivity and catalytic and electrocatalytic activities [21]. Nanostructured materials have great applications in drug analysis in various forms and in biological fluids [22–24]. Modification of electrode surfaces with nanostructured materials has been largely performed in various field of nanomedicine [25–27]. This causes the sensitivity of the measurements to be improved through increments in the active surface area, novel physical properties or unique chemical activities and reactivities of nanosized materials [20–28]. In this regards, gold nanostructures

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20 mmol L−1 HAuCl4 + 0.5 mol L−1 H2 SO4 + 150 mmol L−1 lysine at the potential of 0.0 mV for 600 s. Au/Au-NA electrode was then rinsed thoroughly with distilled water. 2.4. General procedures The real surface areas of the Au and Au/Au-NA electrodes were determined electrochemically using K4 [Fe(CN)6 ] (0.5 mmol L−1 ) as a redox probe. For a reversible redox process, the dependency of the peak current on the potential sweep rate is produced by [32]: Ip = (2.69 × 105)n3/2 AC ∗ D1/2 1/2

Scheme 1. Chemical structure of azathioprine.

have been largely employed in biosensing, imaging, drug delivery, diagnosis and therapy, and antibacterial applications [29–31]. In the present study, electrodeposition of gold in the presence of lysine resulted in the formation of hierarchical dendrites of pyramidal nanoparticles. The nanostructure was then applied to the electrocatalytic reduction and determination of azathioprine. 2. Experimental 2.1. Materials All chemicals were of analytical grade form Merck (Germany) or Sigma (USA) and were used without further purification. Azathioprine was received from Arasto Pharmaceutical Chemicals Inc., Tehran, Iran. The azathioprine tablets were obtained from a local drugstore. All solutions were prepared with doubly distilled water. 2.2. Apparatus Electrochemical experiments were carried out in a conventional three-electrode cell containing a supporting electrolyte powered by a ␮-Autolab type III potentiostat/galvanostat (The Netherlands). A Ag/AgCl, saturated solution of KCl, a glassy carbon rod, and a bare (Au) or modified gold disk electrode with hierarchical nanostructure consisting of an array of parallel arranged pyramidal nanoparticles (Au/Au-NA) were used as the reference, counter and working electrodes, respectively. The system was run on a PC through GPES 4.9 software. In order to obtain information about the morphology and size of the electrodeposited gold nanostructures, field emission scanning electron microscopy (FESEM) was performed using Zeiss, SigmaIGMA/VP instrument (Germany). 2.3. Modification of Au electrode with an array of parallel arranged pyramidal nanoparticles Potentiostatic electrodeposition method was employed to synthesize the gold nanostructure and to prepare Au/Au-NA. Before electrodeposition, the Au electrode was polished by sand papers and then on a polishing pad with 50 nm-alumina powder lubricated by glycerin. Polishing was continued to attain a mirror-like surface. The electrode was then cleaned by immersion in a 1:3 water/ethanol mixture and ultrasonication for 5 min in an ultrasound bath. The electrode was further electropolished by immersion in a 500 mmol L−1 H2 SO4 solution and applying potential in the range of cathodic and anodic edges of the electrolyte stability in a regime of cyclic voltammetry for 25 consecutive cycles. Upon this pretreatment, clean and stable Au electrode surface was attained. The Au electrode was then placed in the cell containing

(1)

where Ip is the peak current, n is the number of exchanged electrons, A is the surface area, C* is the bulk concentration, D is the diffusion coefficient, and  is the potential sweep rate. For the redox transition of [Fe(CN)6 ]4− , n = 1 and D = 7.60 × 10−6 cm s−1 [33]. Cyclic voltammograms using both Au and Au/Au-NA electrodes were recorded and the real surface areas were obtained. Evidently, the Au/Au-NA electrode had a surface area of ∼1.45 times of the Au electrode surface. Standard solutions of azathioprine were prepared by dissolving the drug in a small volume of ethanol, and stored in the dark at 4 ◦ C (ethanol is not electroreactive on the gold electrode in the potential range of electroreactivity of azathioprine. Additional dilutions were performed daily just before use with 100 mmol L−1 sodium phosphate buffer solution, pH 7.40 (PBS). The drug solutions were stable and their concentrations did not change with time. For pH adjustment at different values, appropriate volumes of hydrochloric acid or sodium hydroxide solutions (100 mmol L−1 ) were added to PBS. In all the studies of the drug, PBS was purged from oxygen by introducing a nitrogen (>99.99% purity) stream into the solution for up to 60 min. Degassed PBS was used as the supporting electrolyte throughout the studies on azathioprine. The calibration curve for the drug in PBS was measured with differential pulse voltammetry (DPV). For DPV, a pulse width of 25 mV, a pulse time of 50 ms, and a scan rate of 10 mV s−1 were employed. In order to analyze the drug tablets, the average mass of 10 tablets from each sample was measured. The tablets were then finely powdered and homogenized in a mortar and an accurately weighed amount of this sample was dissolved in an appropriate volume of ethanol (96% v/v). The mixture was then sonicated in an ultrasound bath for 10 min. The supernatant was removed and the clear yellow solution was employed as a stock solution. Appropriate volumes of this solution were diluted with BPS and directly analyzed. 3. Results and discussion Fig. 1 shows FE-SEM images of the Au/Au-NA electrode surface with different magnifications. At low magnifications, FE-SEM images revealed that the surface is covered by long dendrites of ∼1 to 12 ␮m with a rough geometric shape. At higher magnifications, FE-SEM images indicate that each dendrite is a hierarchical nanostructure consisting of an array of parallelly arranged pyramidal nanoparticles of about 0.25 ␮m2 . Typical cyclic voltammograms of Au and Au/Au-NA electrodes recorded in PBS in the absence and presence of 1.0 mmol L−1 azathioprine are shown in Fig. 2. Azathioprine is electroreduced as one cathodic peak in the voltammograms using both the Au and Au/Au-NA electrodes. In adition, no anodic peak appeared in the backward sweep in the voltammograms. This indicates that the electroreduction of azathioprine is irreversible on both the electrode surfaces. The cathodic peak potential and current using the Au electrode are −712 mV and 9.3 ␮A, respectively, while these values

R. Dehdari Vais et al. / Sensors and Actuators B 215 (2015) 113–118

115

2 0 -2 -4

Au/Au-NA-base Au/Au-NA-Aza Au-base Au-Aza

I/ A

-6 -8 -10 -12 -14 -16 -18 -1000

-800

-600

-400

-200

0

E / mV Fig. 2. Typical cyclic voltammograms of Au and Au/Au-NA electrodes recorded in PBS in the absence and presence of 1.0 mmol L−1 azathioprine. The potential sweep rate was 50 mV s−1 .

Fig. 1. FE-SEM images of the Au/Au-NA electrode surface with different magnifications.

for the Au/Au-NA electrode are −605 mV and 15.7 ␮A. The results indicated that azathioprine was reduced on the Au/Au-NA electrode surface at less negative potentials (about 107 mV). Therefore, gold hierarchical dendrites enhanced the reduction process from the thermodynamic point of view. At the same time, the anodic peak current for azathioprine electroreduction using the Au/AuNA electrode was ∼1.7 times higher than that of the Au electrode. Regarding the ratio of the real surface areas of the electrodes (1.45), it is revealed that the gold hierarchical dendrites increased the rate of the reaction more than what is expected from increment in the real surface area of the Au/Au-NA electrode. Gold hierarchical dendrites enhanced the azathioprine electroreduction from both the thermodynamics and kinetics points of view. This will lead to electroreduction of azathioprine to occur at lower potentials with an enhanced sensitivity when the Au/Au-NA electrode is employed for determination of azathioprine. It has been reported that the activity and reactivity of gold depend on its shape and aspect ratio [34–36]. Gold hierarchical dendrites of pyramidal nanoparticles are highly anisotropic structure and can represent high chemical (re)activity, because gold atoms located at the edges have a higher activity. Cyclic voltammograms of electroreduction of 1.0 mmol L−1 azathioprine on the Au/Au-NA electrode surface at different pH values were recorded. The dependencies of the peak potential and current of azathioprine electroreduction on the solution pH derived from these cyclic voltammograms are shown in Fig. 3. Upon increasing the solution pH, the peak current decreased up to pH ∼ 6.5 and then increased. However, because this study is focused on the determination of azathioprine in physiological conditions, pH = 7.40 was chosen as the working pH throughout the work. In addition, the peak potential shifted linearly to more negative values upon increasing the solution pH up to ∼11.8, and then shifted to less negative values. From the slope of the linear dependency of the peak potential on the solution pH, it can be deduced that azathioprine was reduced on the Au/Au-NA electrode surface through a process involving the same electron and proton. Regarding the electroreduction reaction of azathioprine on the Au/Au-NA electrode, azathioprine bears one functional group of nitro as a reducible group. Because a single cathode peak appeared in the voltammograms, based on previous studies on the electroreduction of tertiary nitroalkanes, and the results of study of the effect of pH on electroreduction of azathioprine, the electroreduction reaction is the four-electron four-proton reduction of nitroalkane to the corresponding alkaylhydroxylamine [37], as shown in Scheme 2.

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60

0

-60

-1 -2

-180 y = -56.58x - 197.42 2 R = 0.9974

-300

-3 -4

-420

-5

-540

Epc = (RT/2˛F) ln 

Ip / A

Ep / mV

of the electroreduction process. The electron transfer coefficient of the electroreduction reaction can be obtained using the dependency of the cathodic peak current on the natural logarithm of the potential sweep rate using the following equation [38]:

where Epc , ˛ and  are the anodic peak potential, electron transfer coefficient and the potential sweep rate, and the other variables have their usual meanings. Fig. 4B shows the linear dependency of Epc on ln  for  ≥ 200 mV s−1 , and the electron transfer coefficient was obtained as 0.47. In addition, the heterogeneous electron transfer rate constant and the electron transfer coefficient can also be obtained using the dependency of the natural logarithm of the  cathodic peak current on (Epc −E0 ) values using the following equation [32]:

-6

-660

-7

-780

-8

-900

-9 0

2

4

6

8

10

12

14

pH Fig. 3. The dependencies of the peak potential and current of azathioprine electroreduction on the solution pH. The data was obtained from cyclic voltammograms of electroreduction of 1.0 mmol L−1 azathioprine on the Au/Au-NA electrode surface at different pH levels with a potential sweep rate of 50 mV s−1 .

O

N

N

+

N

N

O

S

S

N N

NHOH

N

+ 4H+ + 4e

N N N

N

N N



Ipc = 0.227FAC ∗ k0 exp[−(˛F/RT )(Epc − E 0 )] C* ,

Fig. 4A shows cyclic voltammograms of 0.5 mmol L−1 azathioprine solution recorded at different potential sweep rates from 5 to 1000 mV s−1 . The peak current increased with increase in the potential sweep rate, and the peak potential shifted to more negative potentials. The latter further confirmed the irreversible nature

and are the cathodic peak current, the where Ipc , n, A, number of exchanged electrons, the electrode surface area, the bulk concentration of azathioprine, the standard rate constant, and the formal potential. The formal potential was obtained as the extrapolated peak potential at potential sweep rate of zero. Fig. 4C shows  the linear dependency of ln Ipc on (Epc −E0 ), and based on this plot, the electron transfer coefficient and the standard rate constant were obtained as 0.46 and 0.014 cm s−1 , respectively. Also, the peak currents in the voltammograms presented in Fig. 4A depend linearly on the square root of the potential sweep rate, as shown in Fig. 4D. Using the Randles–Sevcik equation [32]: (4)

where D is the diffusion coefficient of the drug and the slope of the line presented in Fig. 4D. The diffusion coefficient of azathioprine was obtained as 6.30 × 10−6 cm2 s−1 . To develop a sensitive analytical method for the analysis of azathioprine, DPV was employed. Differential pulse voltammograms of different azathioprine concentrations recorded using the Au/AuNA electrode are shown in Fig. 5 and the corresponding calibration

A

-3

-683 -690

-8

Epc / mV

I / mA

(3)

 E0

k0

Ipc = (2.99 × 105 )˛1/2 n3/2 AC ∗ D1/2 1/2

Scheme 2. The electroreduction reaction of azathioprine.

2

(2)

-13

y = -27.076x - 920.17 R2 = 0.9991

-697 -704 -711 -718

B -725 -8.6

-18

-750

31 29 27 25 23 21 19 17 C 15 -100

-650

-550 -450 E / mV

-350

y = -0.4017x - 10.925 R2 = 0.9886

-93

-86 -79 Epc-E0' / mV

-72

-65

-7.8 ln ( / mV s-1)

-7.4

-250

Ipc / A

Ipc / A

-23 -850

-8.2

-19.5 -20 -20.5 -21 -21.5 -22 -22.5 D -23 0.01

y = -263.4x - 16.135 R2 = 0.995

0.015

0.02

0.025

0.03

( / mV s-1)1/2

Fig. 4. (A) Cyclic voltammograms of 0.5 mmol L−1 azathioprine solution recorded at different potential sweep rates of 5, 7, 10, 20, 40, 75, 100, 150, 200, 250, 300, 350, 400, 450 and 500 mV s−1 . (B) Dependency of the cathodic peak potential on the natural logarithm of the potential sweep rate for  ≥ 200 mV s−1 . (C) Dependency of the natural  logarithm of the cathodic peak current on (Epc −E0 ) for  ≥ 200 mV s−1 . (D) Dependency of the cathodic peak currents in the voltammograms presented in (A) on the square root of the potential sweep rate.

R. Dehdari Vais et al. / Sensors and Actuators B 215 (2015) 113–118 Table 3 Determination of azathioprine in commercial tablets.

0 -1 5 4 Ip / A

I/ A

-2 -3

y = 0.0043x + 0.0537 R2 = 0.9993

3

Tablet

Amount labeled, mg

Amount found, %w/w

Bias%

A B C

50 50 50

48.3 49.0 48.5

−3.4 −2.0 −3.0

2 1

-4

0 200

0

-5

400

600

C / mol L

-6 -750

-650

-550

-450

-350 E / mV

-250

800

1000

-1

-150

-50

50

0.33 0.28 Ip / A

117

0.23 0.18 0.13

0.06

0.08

0.055

0

20 C / mol L

40

60

-1

Ip / A

0.03

0.05

the same line with the reported values. The values of the analytical parameters obtained for the azathioprine tablets according to this method are reported in Table 3. Selectivity of DPV responses for the azathioprine assay was examined in the presence of some chemicals. The results showed no significant interference from Al(III), Ca(II), Co(II), Cr(III), Mg(II), Mn(II), Ni(II), Zn(II), ascorbic acid, uric acid, alanine, tyrosine, dopamine, glucose and sucrose, at least for the same concentrations of the drugs and these chemicals. These compounds are not electroreactive toward the electroreduction in the working potential range. Therefore, the procedure is able to do the azathioprine assay with considerable selectivity.

0.045 0.04

4. Conclusion

0.035 0.03

0

1

2

3

C / mol L

4

-1

Fig. 5. Differential pulse voltammograms of different azathioprine concentrations. Inset: The corresponding calibration curve. Table 1 The determined parameters for the calibration curve of azathioprine and accuracy and precision using the Au/Au-NA electrode. Linear range, ␮mol L−1 Sensitivity (slope), A L mol−1 cm−2 Intercept, ␮A cm−2 R2 Standard error of slope (P = 0.005) Standard error of intercept (P = 0.005) LOD, ␮mol L−1 LOQ, ␮mol L−1 RSD%

0.095–900 0.14 1.71 0.9993 3.56 × 10−5 0.01 0.027 0.090 3.85

curve is shown in the inset. The limits of detection (LOD) and quantitation (LOQ) of the procedure were calculated according to the 3SD/m and 10SD/m criteria respectively, where SD is the standard deviation of the intercept and m is the slope of the calibration curve [39]. The determined parameters for the calibration curve of azathioprine based on the DPV measurements are represented in Table 1. A comparison between the analytical parameters of some methods for determination of azathioprine was made, as displayed in Table 2. The applicability of the proposed DPV method for the sample dosage form was examined by analyzing the azathioprine tablets. It was found that the matrices of the tablet did not show any interference and the drug amounts determined using this method are in Table 2 A comparison between the analytical parameters of some methods for the determination of azathioprine. Method

Linear range, ␮mol L−1

LOD, ␮mol L−1

Reference

Spectrophotometry HPLC HPLC Capillary zone electrophoresis HPLC DPV

3.61–28.85 7.21–79.4 – 36.1–180.3

0.087 0.32 0.018 2.71

[11] [13] [14] [18]

0.28–3.60 0.095–900

0.30 0.090

[40] This work

A green electrodeposition method was developed for the synthesis of gold hierarchical dendrites of parallel-arranged pyramidal nanoparticles on a gold electrode surface, and then employed to fabricate an azathioprine sensor. The synthesis method can be extended to the synthesis of other gold nanostructures or other noble metal nanostructures with changes in the synthesis parameters. The hierarchical dendrites showed an efficient activity toward the electroreduction of azathioprine in physiological medium resulting from special size, shape and structure of the dendrites. Voltammetric responses generated by the dendrites confirmed that the drug can be determined by high sensitivity in commercial tablets. The electrode can be applied in routine analysis of the drug, having the potential to be used for successful determination of azathioprine in pharmaceutical preparations. Acknowledgments We would like to thank the Research Council of Shiraz University of Medical Sciences (7930) and also the Iran National Science Foundation (INSF) for supporting this research. References [1] J.S. Maltzman, G.A. Koretzky, Azathioprine: old drug, new actions, J. Clin. Invest. 111 (2003) 1122–1124. [2] M.E. Schram, R.J. Borgonjen, C.M. Bik, J.G. Schroeff, J.J. Everdingen, P.I. Spuls, Offlabel use of azathioprine in dermatology a systematic review, Arch. Dermatol. 147 (2011) 474–488. [3] J.K. Marshall, Review: azathioprine, infliximab, certolizumab, and adalimumab are effective for maintaining remission in crohn’s disease, Evid. Based Med. 13 (2008) 115. [4] J.C. Berridge, Pharmaceutical analysis in a multidisciplinary development environment, J. Pharm. Pharmacol. 45 (1993) 361–366. [5] J. Chamberlain, The Analysis of Drugs in Biological Fluids, second ed., CRC Press, Boca Raton, Florida, USA, 1995. [6] V.K. Gupta, R. Jain, K. Radhapyari, N. Jadon, S. Agarwal, Voltammetric techniques for the assay of pharmaceuticals – a review, Anal. Biochem. 408 (2011) 179–196. [7] A. Cringauz, Introduction to Medicinal Chemistry: How Drugs Act and Why, Wiley-VCH, New York, 1997. [8] R.L. Ruth, T.W. Carol, D.S. Rochelle, Pharmacology: Drug Actions and Reactions, fifth ed., The Parthenon Publishing Group, International Publishers in Medicine, Science and Technology, 1996. [9] Q. Xu, A.J. Yuan, R. Zhang, X.J. Bian, D. Chen, X.Y. Hu, Application of electrochemical methods for pharmaceutical and drug analysis, Curr. Pharm. Anal. 5 (2009) 144–155. [10] S.A. Ozkan, B. Uslu, H.Y. Aboul-Enein, Analysis of pharmaceuticals and biological fluids using modern electroanalytical techniques, Crit. Rev. Anal. Chem. 33 (2003) 155–181.

118

R. Dehdari Vais et al. / Sensors and Actuators B 215 (2015) 113–118

[11] C.S. Lakshmi, M.N. Reddy, Spectrophotometric determination of azathioprine in pharmaceutical formulations, Talanta 47 (1998) 1279–1286. [12] J.B. Wang, P. Zhao, S.Q. Han, Direct determination of azathioprine in human fluids and pharmaceutical formulation using flow injection chemiluminescence analysis, J. Chin. Chem. Soc. 59 (2012) 239–244. [13] T.T. Fazio, A.K. Singh, E.R.M. Kedor-Hackmann, M.I.R.M. Santoro, Quantitative determination and sampling of azathioprine residues for cleaning validation in production area, J. Pharm. Biomed. Anal. 43 (2007) 1495–1498. [14] E.C. Van Os, J.A. Kinney, B.J. Zins, D.C. Mays, Z.H. Schriver, W.J. Sandborn, J.J. Lipsky, Simultaneous determination of azathioprine and 6-mercaptopurine by high-performance liquid chromatography, J. Chromatogr. B: Biomed. Sci. Appl. 679 (1996) 1–2. [15] N.G. Goger, H.K. Parlatan, H. Basan, A. Berkkan, T. Ozden, Quantitative determination of azathioprine in tablets by H-1 NMR spectroscopy, J. Pharm. Biomed. Anal. 21 (1999) 685–689. [16] S. Kulkarni, K. Chitalkar, N. Shinde, P. Tekale, R. Lanjewar, A validated highperformance thin-layer chromatographic method for the determination of azathioprine from pharmaceutical formulation, JPC: J. Planar Chromat.-Mod. TLC 27 (2014) 120–123. [17] P.M. Davadra, V.V. Mepal, M.R. Jain, C.G. Joshi, A.H. Bapodra, A validated UPLC method for the determination of process related impurities in azathioprine bulk drug, Anal. Methods 3 (2011) 198–204. [18] A. Shafaati, B.J. Clark, Determination of azathioprine and its related substances by capillary zone electrophoresis and its application to pharmaceutical dosage forms assay, Drug Dev. Ind. Pharm. 26 (2000) 267–273. [19] S.P. Chen, Z. Qiao, A. Vivoni, C.M. Hosten, Determination of the orientation of azathioprine adsorbed on a silver electrode by SERS and ab initio calculations, Spectrochim. Acta A 59 (2003) 2905–2914. [20] B. Bhushan (Ed.), Springer Handbook of Nanotechnology, third ed., Springer, New York, 2010. [21] I. Malsch, C. Emond (Eds.), Nanotechnology and Human Health, CRC, Press, New York, 2014. [22] H. Heli, N. Sattarahmady, R. Dehdari Vais, K. Karimian, Nickel hydroxide nanopetals: one-pot green synthesis, characterization and application for the electrocatalytic oxidation and sensitive detection of montelukast, Sens. Actuat. B 196 (2014) 631–639. [23] G. Gedda, S. Pandey, M.L. Bhaisare, H.F. Wu, Carbon dots as nanoantennas for anti-inflammatory drug analysis using surface-assisted laser desorption/ionization time of flight mass spectrometry in serum, RSC Adv. 4 (2014) 38027–38033. [24] M. Regiart, S.V. Pereira, V.G. Spotorno, F.A. Bertolino, J. Raba, Nanostructured voltammetric sensor for ultra-trace anabolic drug determination in food safety field, Sens. Actuators B: Chem. 188 (2013) 1241–1249. [25] T. Berthing, C.B. Sørensen, J. Nygard, K.L. Martinez, Applications of nanowire arrays in nanomedicine, J. Nanoneurosci. 1 (2009) 3–9. [26] J.E.N. Dolatabadi, O. Mashinchian, B. Ayoubi, A.A. Jamali, A. Mobed, D. Losic, Y. Omidi, M. Guardia, Optical and electrochemical DNA nanobiosensors, Trends Anal. Chem. 30 (2011) 459–472. [27] F. Valentini, M. Carbone, G. Palleschi, Carbon nanostructured materials for applications in nanomedicine, cultural heritage, and electrochemical biosensors, Anal. Bioanal. Chem. 405 (2013) 451–465. [28] N. Sattarahmady, H. Heli, R. Dehdari Vais, A flower-like nickel oxide nanostructure: synthesis and application for choline sensing, Talanta 119 (2014) 207–213.

[29] P.K. Jain, X. Huang, I.H. El-Sayed, M.A. El-Sayed, Noble metals on the nanoscale: optical and photothermal properties and some applications in imaging, sensing, biology, and medicine, Acc. Chem. Res. 41 (2008) 1578–1586. [30] X.F. Li, C.Y. Chen, Y.H. Zhao, X.Y. Yuan, Surface modification of gold nanorods and their applications in combination of cancer diagnosis and therapy, Prog. Biochem. Biophys. 41 (2014) 739–748. [31] A.K. Khan, R. Rashid, G. Murtaza, A. Zahra, Gold nanoparticles: synthesis and applications in drug delivery, Trop. J. Pharm. Res. 13 (2014) 1169– 1177. [32] A.J. Bard, L.R. Faulkner, Electrochemical Methods, Wiley, New York, 2001. [33] S.F. Wang, Q. Xu, Electrochemical parameters of ethamsylate at multi walled carbon nanotube modified glassy carbon electrodes, Bioelectrochemistry 70 (2007) 296–300. [34] T.S. Sreeprasad, A.K. Samal, T. Pradeep, Reactivity and resizing of gold nanorods in presence of Cu2+ , Bull. Mater. Sci. 31 (2008) 219–224. [35] S. Hebie, L. Cornu, T.W. Napporn, J. Rousseau, B.K. Kokoh, Insight on the surface structure effect of free gold nanorods on glucose electrooxidation, J. Phys. Chem. C 117 (2013) 9872–9880. [36] N.R. Jana, L. Gearheart, S.O. Obare, C.J. Murphy, Anisotropic chemical reactivity of gold spheroids and nanorods, Langmuir 18 (2002) 922–927. [37] H. Lund, in: H. Lund, O. Hammerich (Eds.), Inorganic Electrochemistry, Marcel Dekker, New York, 1991, pp. 725–764. [38] J.A. Harrison, Z.A. Khan, The oxidation of hydrazine on platinum in acid solution, J. Electroanal. Chem. 28 (1970) 131–138. [39] J.C. Miller, J.N. Miller, Statistics for Analytical Chemistry, fourth ed., EllisHarwood, New York, 1994. [40] T. Pawinski, M. Gajewska, Determination of azathioprine, prednisone and methylprednisolone in human blood serum by high performance liquid chromatography, Chem. Anal. (Warsaw) 42 (1997) 233–238.

Biographies Ms. R. Dehdari Vais received her M.Sc. degree in Analytical Chemistry from Islamic Azad University, Fars, Iran. Her research interests are focused on the Fabrication and characterization of novel nanostructured materials for electroanalytical and medical applications. Dr. N. Sattarahmady received her Ph.D. in biophysics from Institute of Biochemistry and Biophysics, University of Tehran, Iran. She currently is an associate professor of biophysics at Shiraz University of Medical Sciences. Her current interests include protein structure, design of nanostructured materials for encapsulation, drug delivery and biosensors, and nanomedicine. Dr. K. Karimian received his Ph.D. in Bioorganic Chemistry from Louisiana State University, 1976. He currently is Managing Director of Arasto Pharmaceutical Chemicals, Inc., Iran. Dr. H. Heli received his Ph.D. in electrochemistry from K. N. Toosi University of Technology, Tehran, Iran. He currently is an assistant professor of chemistry at Shiraz University of Medical Sciences, Shiraz, Iran. His major interests are synthesis of new nanostructured and targeted materials and their applications in electrocatalysis, bioelectrocatalysis, and electrochemical and medical nano-devices.