Biosensors and Bioelectronics 41 (2013) 608614

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Biosensors and Bioelectronics

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Electrosynthesis of an imidazole derivative and its application as a bifunctional electrocatalyst for simultaneous determination of ascorbic acid, adrenaline, acetaminophen, and tryptophan at a multi-wall carbon nanotubes modied electrode surface
Navid Nasirizadeh a,n, Zahra Shekari a, Hamid R. Zare b, M. Reza Shishehbore c, Ali R. Fakhari d, Hamid Ahmar d
a

Scientic Society of Nanotechnology, Yazd Branch, Islamic Azad University, Yazd, Iran Department of Chemistry, Yazd University, P.O. Box 89195-741, Yazd, Iran Department of Chemistry, Yazd Branch, Islamic Azad University, Yazd, Iran d Department of Chemistry, Faculty of Sciences, Shahid Beheshti University, G. C., P.O. Box 19839-4716, Tehran, Iran
b c

a r t i c l e i n f o
Article history: Received 28 June 2012 Received in revised form 16 September 2012 Accepted 20 September 2012 Available online 27 September 2012 Keywords: Electrosynthesis Bifunctional Ascorbic acid Adrenaline Simultaneous determination

a b s t r a c t
In this research, the electrosynthesis of 4-(1H-benzo[d]imidazol-2-ylthio)-5-methylbenze-1,2-diol (as an imidazole derivative) is reported. An imidazole derivative multi-wall carbon nanotube modied glassy carbon electrode (IMWCNTGCE) was constructed and used as an excellent bifunctional electrocatalyst for oxidation of ascorbic acid (AA) and adrenaline (AD). Cyclic voltammetry was used to calculate the surface electron transfer rate constant, ks, and the electron transfer coefcient, a, for the electron transfer between MWCNT GCE and the electrodeposited imidazole derivative. The kinetic parameters such as the electron transfer coefcient, a, and the heterogeneous rate constant, k, for the oxidation of AA and AD at the IMWCNTGCE surface were estimated. The modied electrode was found quite effective for the simultaneous determination of AA, AD, acetaminophen (AC), and tryptophan (Trp) in a mixture solution. The detection limits of AA and AD were calculated as 0.96 mM and 0.38 mM, respectively. Finally, IMWCNTGCE was satisfactorily used for the determination of AA, AD, and AC in pharmaceutical samples. & 2012 Elsevier B.V. All rights reserved.

1. Introduction Now a days, the development of voltammetric methods for simultaneous determination of biomolecules in human uids has been fascinating to biologists and analytical researchers (Zare and Nasirizadeh, 2011; Shahrokhian et al., 2009; Zare et al., 2006; Zare and Nasirizadeh, 2009). Biochemical compounds such as ascorbic acid (AA), adrenaline (AD), acetaminophen (AC), and tryptophan (Trp) are oxidized at nearly the same potential with poor sensitivity at unmodied electrodes. The overlap of their voltammetric responses makes their simultaneous determination highly difcult (Noroozifar et al., 2011). To overcome this problem, various modied electrodes have been constructed (Zare et al., 2005; Babaei et al., 2008; Nasirizadeh and Zare, 2009; Alothman et al., 2010; Zare and Nasirizadeh, 2010; Habibi et al., 2011). AD and AA always exist together in biological uids, at most solid

Corresponding author. Tel.: 98 351 8117711; fax: 98 351 8117550. E-mail address: nasirizadeh@yahoo.com (N. Nasirizadeh).

electrodes; AA is oxidized at a potential close to that of AD, resulting in an overlapping voltammetric response. Electroanalytical methods with modied electrodes have raised great interest in simultaneous determination of the two compounds (Chen et al., 2006; Ensa et al., 2010; Lu et al., 2011). A complementary presence of AA intensies the main favourable effect of AC and concomitantly compensates for the potential toxicity in the function of the liver (Grundmann et al., 2006). Moreover, much of the corresponding literature points to major interferences between AA and AC (Habibi et al., 2011). Therefore, determining AC and AA simultaneously promises to be of great importance for the illustration of their precise physiological functions. Also, in the presence of AA, Trp can be converted into 5-hydroxytryptophan which forms serotonin, an important brain chemical in animals. Determining Trp and AA concentration in animal blood can help scientists control the production of serotonin in the body (Babaei et al., 2008). As widely known, the oxidation peaks of AA, AD, and AC are nearly at the same potential on a bare electrode, which results in overlapping voltammetric responses, hence making their distinction highly

0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.09.028

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difcult. They usually coexist in the extra cellular uid of the central nervous system and serum. Thus, determining the neurotransmitter AD and its coexisting species individually or simultaneously is an important issue not only in the eld of biomedical chemistry but also in diagnostic and pathological research. To meet the purpose, it is necessary to make further efforts for the fabrication of simple, rapid, selective and sensitive electrochemical modied electrodes that can be used for simultaneously determination of the above-mentioned biomolecules in presence of each other. Electrosynthesis is a safe and low-cost method that is widely used for the synthesis of organic compounds. Catechol and hydroquinone redox systems have been extensively studied because of their biological and environmental importance, as well as the classical fundamental importance of their redox chemistry. There has been a growing interest in the study of reactions involving quinones produced through the oxidation of catechols versus other nucleophiles. This is due to the mechanistic and synthetic importance of these reactions (Fakhari et al., 2007; Shayani-Jam and Nematollahi, 2010; Costentin, 2008; Varmaghani and Nematollahi, 2011). Therefore, we have allocated the present study to the synthesis of 4-(1H-benzo[d]imidazol-2ylthio)-5-methylbenze-1,2-diol (as an another imidazole derivative). The compound has been used to modify multi-wall-carbon nanotubes (MWCNT) deposited at a glassy carbon electrode (GCE) surface. In this study, an imidazole derivative and MWCNT modied glassy carbon electrode (IMWCNTGCE), serving as an excellent bifunctional electrocatalyst, has been used for electrocatalytic oxidation of AA and AD individually and in the presence of AC and Trp. The literature review indicates that there are not any reports about chemically modied electrode that can be used for electrocatalytic oxidation of both species of AA and AD, and simultaneous determination of AA, AD, AC, and Trp.

of the electrolysis, the cell was kept in a refrigerator overnight. The precipitated solid was collected by ltration and washed thoroughly with distillated water. After checking the purity of the resulted material, the product was characterized using IR, 1 H NMR, 13C NMR, and MS (see S1, Supplementary Information). 2.3. Preparation of IMGCE, MWCNT GCE and IMWCNTGCE MWCNT-modied GCE was prepared as following: after mechanical polishing of GCE using 0.05 mm alumina in water slurry using a polishing cloth and rinsing it with double distilled water, the electrochemical activation of the electrode was performed by a continuous potential cycling from 1.45 to 1.7 V at scan rate potential of 100 mV s 1 in a sodium bicarbonate (0.1 M) solution. 3 mL of a homogenized DMFMWCNT mixture (1 mg/5 mL) was placed directly onto the activated GCE surface and dried at room temperature to form a MWCNT lm at the GCE surface. IMWCNTGCE was prepared by modifying the MWCNT GCE in 0.15 M phosphate buffer solution (pH 7.0) containing 0.10 mM of the imidazole derivative by 16 cycles of scan rate potentials between 100 mV and 500 mV at 20 mV s 1. To fabricate the imidazole modied GCE (IMGCE), the activated GCE was placed in a 0.15 M phosphate buffer (pH 7.0) containing 0.10 mM of the imidazole derivative. It was modied with the same procedure described for IMWCNTGCE.

3. Results and discussion 3.1. Electrosynthesis of 4-(1H-benzo[d]imidazol-2-ylthio)5-methylbenze-1,2-diol The cyclic voltammogram of 1.0 mM 4-methylcatechol in a water/acetonitrile (85/15) solution containing a 0.15 M phosphate buffer (pH 7.0) shows an anodic and a corresponding cathodic peak, which correspond to the redox reaction of 4-methylcathecol, the corresponding o-benzoquinone, and vice versa through a quasi-reversible two-electron process (Fig. 1, curve a). A ratio of the cathodic peak current to the anodic peak current (Ipc/Ipa) is near to 1, particularly during the repetitive recycling of the potential. It can be considered as a criterion

2. Experimental 2.1. Apparatus and chemicals A potentiostat (mAutolab, Eco Chemie, Utrecht, Netherlands), was used for electrochemical measurements. The working electrode was IMWCNTGCE, and the reference electrode was a saturated calomel electrode (SCE). Also, a platinum electrode (Azar Electrode Co, Iran) was used as the auxiliary one. The pH measurements were done with a Metrohm model 691 pH/mV meter. The multi-wall carbon nanotubes (1020 nm in diameter, 520 mm long, and 95% pure) were purchased from NanoLab Inc. (Brighton, MA). 4-methylcatechol, 2-mercaptobenzimidazole, phosphate salts, silica gel 60 (0.2 0.5 mm), AA, AD, AC, Trp, and the other reagents were provided from Merck Company and used as received. An injection solution of AD and vitamin C, tablets of AC, and an oral solution of AC were purchased in a local drugstore. 2.2. Electro-organic synthesis of an imidazole derivative In a typical procedure, a 100-mL mixture of water/acetonitrile (85/15) containing phosphates (KH2PO4/K2HPO4) as the buffer and supporting electrolyte (pH 7.0, C 0.15 M) was preelectrolyzed at a chosen potential (0.15 V) in an undivided cell. Subsequently, 2 mmolof catechol and 2 mmol of nucleophile were added to the cell, and electrolysis was performed at the same potentials. The electrolysis was terminated once and more than 95% of the primary current was decayed. The process was interrupted several times during the electrolysis, and the carbon anode was washed in acetone in order to reactivate it. At the end

65
a

25 I / A

-15

-55 -0.5

0 0.5 E / V vs. Ag/AgCl/KCl (sat.)

Fig. 1. Cyclic voltammograms of (a) 1 mM of 4-methylcatechol, (b) 1 mM of 4-methylcatechol in the presence of 1 mM of 2-mercaptobenzimidazole and (c) 1 mM of 2-mercaptobenzimidazole at a glassy carbon electrode in phosphate buffer (pH 7.0, 0.15 M); scan rate potential of 100 mV s 1 and room temperature.

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for the stability of o-benzoquinone produced at the surface of the electrode under experimental conditions. The electrochemical oxidation of 4-methylcathecol was then studied in the presence of 2-mercaptobenzimidazole as a nucleophile. Fig. 1, curve b, shows the cyclic voltammogram obtained from a 1.0 mM 4-methylcatechol in the presence of 1.0 mM 2mercaptobenzimidazole. For a comparison, the cyclic voltammogram of 1.0 mM of 2-mercaptobenzimidazole is shown in Fig. 1, curve c. The multi-cyclic voltammograms of 4-methylcatechol in the presence of 2-mercaptobenzimidazole exhibit a relatively intense decrease in the anodic peak together with some potential shift in a positive direction. The positive shift of the anodic peak in the presence of 2-mercaptobenzimidazole is due to the formation of a thin lm of the product at the surface of the electrode in experimental conditions (Fig. S1) (Bard and Faulkner, 2001). The voltammograms of 1.0 mM 4-methylcatechol in the presence of 1.0 mM 2-mercaptobenzimidazole at various potential scan rates are shown in Fig. S2. It can be observed that the height of the cathodic peak increases in accordance with the augmentation of the potential scan rate (Fig. S2, curves a h). The anodic peak relates to the oxidation of 2-mercaptobenzimidazole. Moreover, the ratio of peak current (IpcI/IpaI) slightly increases with increasing of potential scan rate (Fig. S2, curve i), that shows a chemical reaction following the electron transfer step. Controlled potential coulometry was also performed in a water/acetonitrile (85/15) solution containing 0.25 mM of 4-methylcatechol and 0.25 mM of

2-mercaptobenzimidazole. Considering the proximity of oxidation potential peaks of 1 and c (Fig. 1), to minimize the oxidation of 2-mercaptobenzimidazole and, hence, to achieve a higher selectivity, we applied 0.15 V potential versus Ag/AgCl/KCl (sat.) in both coulometry and preparative synthesis processes. Monitoring the electrolysis progress was carried out by cyclic voltammetry (Fig. S3). The anodic peak decreased and disappeared when the charge consumption became about 2e per molecule of 4-methylcatechol. The voltammetry, coulometry and spectral data allow us to propose an EC mechanism (Fakhari et al., 2008) for the electrooxidation of catechol in the presence of 2-mercaptobenzimidazole (Scheme S1). 3.2. Electrochemical behavior of IMWCNTGCE The cyclic voltammograms of IMWCNTCCE in 0.15 M phosphate buffer solution (pH 7.0) at various potential scan rates are shown in Fig. 2A. As it can be seen, two well dened redox couples corresponding to the electrodeposited imidazole derivative appear when the potential scan is performed oin the range 100 mV300 mV. Insets a and b of Fig. 2A show the plots of the anodic and cathodic peak currents as a function of a potential scan rate that is linear as predicted theoretically for a surfaceimmobilized redox couple. The variations of the peak potential versus the logarithm of the potential scan rate were shown in Figs. 2B and D for redox pair I

0.17 EpI / V 0.08 -0.01 -0.1 0.5

4.5

1.6

2.7

3.8

log (v / mV s-1) 0.33 1.5 EpI / V 0.22 0.11 0 2.9 3.2 3.5 3.8

I / A

log (v / mV s-1) 0.36 -1.5 EpII / V 0.24 0.12 0 0.4 1.6 2.8 4

log (v / mV s-1) 0.18 -4.5 -2.0 EpII / V 0.08 0.36 E / V vs. Ag/AgCl/KCl (sat.) 0.64 0.08 -0.02 -0.12 2.85 3.1 3.35 3.6

log (v / mV s-1)
Fig. 2. (A) Cyclic voltammetric responses of the IMWCNTGCE in 0.15 M phosphate buffer solution (pH 7.0) at different scan rates potential. The numbers 120 correspond to 5100 mV s 1, respectively. Insets: Plots of anodic and cathodic peak currents vs. the scan rate potential for (a) redox pair of I and (b) redox pair of II. (B,D) Variation of the peak potentials vs. the logarithm of the scan rate potential for redox pair of I and II, respectively. (C,E) Magnication of the plot B and D for high scan rates potential.

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and II, respectively. At high potential scan rates, the separation between peak potentials increases with increasing potential scan rate (Figs. 2B and D) that conrms the limitation arising from charge transfer kinetic. The results show that the values of the anodic and cathodic peak potentials are proportional to the logarithm of the potential scan rate at scan rates higher than 800 mV s 1 for redox pair I (Fig. 2C) and 1000 mV s 1 for redox pair II (Fig. 2E). Under these conditions, the surface electron transfer rate constant, ks, and the electron transfer coefcient, a, for the electron transfer between the imidazole derivative and MWCNT GCE can be estimated from the linear variation of the oxidation and reduction peak potentials with the logarithm of potential scan rate according to the Laviron theory for both redox pairs I and II (Laviron, 1979). For high potential scan rates, this theory predicts a linear dependence of Ep upon log v, which can be used to extract the kinetic parameter a from the slope of such plots. Using these plots at pH 7.0, the values of a 0.46, ks 13.45 7 0.39 and a 0.47, ks 20.87 7 0.57 were obtained for redox pairs I and II, respectively. 3.3. Electrocatalytic oxidation of AD and AA at IMWCNTGCE Fig. 3A shows the cyclic voltammograms of IMWCNTGCE in a buffer solution, pH 7.0, in the absence (voltammogram a) and the

presence of 0.40 mM (voltammogram b) AA. There is a drastic enhancement of anodic peak current I but virtually no current in the cathodic sweep. Also, the cyclic voltammetric responses of the electrochemical oxidation of 0.40 mM AA at IMGCE (curve d), MWCNT GCE (curve e), and activated GCE (curve f) are shown in Fig. 3A. As it can be seen, the anodic peak potential for the oxidation of AA at IMWCNTGCE (curve b) and IMGCE (curve d) is about 25 mV, while at MWCNT GCE (curve e) and the activated GCE (curve f), the peak potentials are about 71 and 92 mV, respectively. Thus, a decrease occurs in the overpotential and enhancement peak current for AA at the modied electrode surface. These behaviors point to a strong electrocatalytic effect. The electrocatalytic oxidation of AD at IMWCNTGCE is shown in Fig. 3B. As the results suggest, there is a similar behavior for the oxidation of AD. For example, the electrocatalytic anodic peak potential of AD appears at 180 mV at IMWCNTGCE (Fig. 3B, voltammogram b), while at MWCNT GCE (curve e) and the activated GCE (curve e) the oxidation currents are observed at about 370 and 198 mV, respectively. The electrocatalytic oxidation characteristics of AA and AD at various modied electrode surfaces at pH 7.0 are given in Table 1. Fig. 3C shows the cyclic voltammograms of IMWCNTGCE in a buffer solution, pH 7.0, in the absence (voltammogram a) and presence of 0.20 mM AA and 0.10 mM AD (voltammogram b). Therefore, it is possible to individually or

2 b e b e 1.1 f I / A a c 0.2 d f

1.8 I / A

d 0.6 a c

-0.6 -0.12

-0.02

0.08

0.18

-0.7 0.02

0.13

0.24

0.35

E / V vs. Ag/AgCl/KCl (sat.) 2.4

E / V vs. Ag/AgCl/KCl (sat.)

b 1.4 I / A

0.4

II

-0.6 -0.16

0.01

0.18

0.35

E / V vs. Ag/AgCl/KCl (sat.)

Fig. 3. (A) Cyclic voltammograms of IMWCNTGCE in 0.15 M phosphate buffer solution (pH 7.0) in the absence (a) and presence of 0.40 mM AA (b). (c) as (a) and (d) as (b) in IMGCE. (e, f) as (b) for MWCNT modied and activated GCE. (B) Cyclic voltammograms of IMWCNTGCE in 0.15 M phosphate buffer solution (pH 7.0) in the absence (a) and presence of 0.20 mM AD (b). (c) as (a) and (d) as (b) in IMGCE. (e, f) aAs (b) for MWCNT modied and activated GCE. (C) Cyclic voltammograms of IMWCNTGCE in the buffer solution, pH 7.0, in the absence (a) and the presence of 0.20 mM AA and 0.10 mM AD (b) (scan rate potential 20 mV s 1).

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Table 1 Comparison of electrocatalytic oxidation of AA (0.4 mM) and AD (0.2 mM) on various electrode surfaces at pH 7.0. Name of electrode Oxidation potential (mV) AA AGCE IMGCE MWCNT GCE IMWCNTGCE 92 25 71 25 AD 217 183 195 181 Oxidation current (mA) AA 0.32 0.82 1.28 1.33 AD 0.95 0.83 1.23 1.39

IMGCE, imidazole modied glassy carbon electrode; MWCNT GCE, multi-wall carbon nanotubes modied glassy carbon electrode; IMWCNTGCE, imidazole multi-wall carbon nanotubes modied glassy carbon electrode.

simultaneously determine AA and AD in mixture samples at IMWCNTGCE without any interference. The cyclic voltammograms of the 0.15 M phosphate buffer solution (pH 7.0) containing 0.30 mM AA and 0.10 mM AD at different potential scan rates are recorded (Figs. S4 and S5). From these voltammograms, the approximate values for the total number of electrons involved in the anodic oxidation of AA and AD emerge to be n 1.94 2 and n 1.93 2, respectively. Also, the values of j0 for AA and AD are found to be 25.3 7 0.5 and 3.98 7 0.11 mA cm 2 (see S2, Supplementary information, Figs. S4 and S5). Moreover, Figs. S4 and S5 show that by increasing the potential scan rate, the plot of the peak current versus the square root of potential scan rate deviates from linearity, suggesting a

9 14.5 Ip / A 6.5 4 1.5 550

1250 1950 [AA] / M

2650

8.1

10

Ip / A

6.1

4.1

I / A

2.1 90

210 [AD] / M

330

450

9 6 3 0 110

5.5

Ip/ A

250 [AC] / M

390

530

12

Ip / A 1 -0.2 0.15 0.5 0.85 E / V vs. Ag/AgCl/KCl (sat.)

0 150

400 [Trp] / M

650

900

Fig. 4. (A) Differential pulse voltammograms of IMWCNTGCE in a 0.15 M phosphate buffer solution (pH 7.0) containing different concentrations of AA, AD, AC and Trp. Numbers 117 correspond to 681.82500.0 mM AA, 113.6416.7 mM AD, 136.4500.0 mM AC and 227.3833.3 mM Trp. Inset shows DPV of a mixed solution of 1500.0 mM AA, 300.0 mM AD, 400.0 mM AC and 700.0 mM Trp at a bare GCE. (B)(E) show the plots of the electrocatalytic peak current as a function of AA, AD, AC and Trp concentrations, respectively.

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Table 2 Determination and recovery results of AA and AD in an injection solution, oral solution of AC and tablet of AA and AC at IMWCNTGCE. Samples Added (mM) Found (mM) RSD % Recovery %

Injection solution of AA AA 500.0 AD 150.0 AC 150.0 Trp 250.0 Injection solution of AD AA 750.0 AD 75.0 AC 200.0 Trp 300.0 Oral solution of AC AA 1000.0 AD 200.0 AC 100.0 Trp 250.0 Tablet of AA AA 500.0 AD 150.0 AC 150.0 Trp 300.0 Tablet of AC AA 750.0 AD 150.0 AC 100.0 Trp 250.0

1000.0 250.0 300.0 500.0 1500.0 150.0 300.0 450.0 1500.0 300.0 200.0 500.0 1000.0 250.0 300.0 500.0 1500.0 300.0 200.0 500.0

1500.0 350.0 450.0 750.0 2250.0 225.0 400.0 600.0 2000.0 400.0 300.0 750.0 1500 300.0 450.0 700.0 2250.0 450.0 300.0 750.0

743.0

1246.0 146.0 152.0 256.0 744.0 234.0 196.0 309.0 983.0 203.0 299.0 246.0 1325.0 147.0 154.0 292.0 746.0 151.0 301.0 246

1739.0 254.0 307.0 488.0 1477.0 304.0 307.0 455.0 1491.0 294.0 391.0 488.0 1812.0 257.0 304.0 512.0 1489.0 294.0 404.0 488

2261.0 358.0 439.0 751.0 2269.0 380.0 395.0 584.0 2019.0 409.0 491.0 761.0 2290.0 297.0 441.0 711.0 2273.0 455.0 498.0 763.0

1.2 1.7 2.0 1.2 2.5

1.9 2.3 3.0 1.8 2.5 1.9 2.1 2.4 2.2 2.7 3.1 1.7 1.9 2.1 2.4 2.3 2.3 1.8 2.2 1.9

2.9 1.5 1.6 2.1 1.3 2.3 2.0 1.9 1.5 1.3 1.8 2.4 2.3 2.6 2.9 1.8 2.0 2.8 1.7 2.4

2.7 2.0 2.4 2.8 1.8 2.8 1.6 2.1 1.9 2.2 2.1 2.5 1.5 1.8 2.8 1.6 1.9 3.0 2.4 2.9

100.6 97.3 101.3 102.4 99.2 101.3 98.0 103.0 98.3 101.5 103.0 98.4 100.8 98.0 102.0 97.3 99.5 100.7 98.0 98.4

99.6 101.6 102.3 97.6 98.5 97.3 102.3 101.1 99.4 98.0 97.5 97.6 99.1 102.8 101.3 102.4 99.3 98.0 100.5 97.6

101.2 102.3 97.6 100.1 100.8 98.7 101.3 97.3 100.9 102.2 98.3 101.5 97.9 99.0 98.0 101.6 101.0 101.1 98.3 101.7

158.0 196.0 821.0 203.0

kinetic limitation in the reaction of the oxidized form of imidazole and the analytes (AA and AD). Based on these results, a catalytic reaction mechanism (ErCi) describes the oxidation reaction of the analyte by the imidazole derivative. Under the above conditions for ErCi mechanisms, the Andrieux and Saveant (1978) theoretical model can be used to calculate the catalytic rate constant. Using the theoretical paper by Andrieux and Saveant, the average values of k (4.96 7 0.04) 10 4 cm s 1 and k (1.17 7 0.04) 10 3 cm s 1 are obtained for AA and AD, respectively. These values of k explain a good catalytic feature for the oxidation of AA and AD at IMWCNTGCE (see S2, Supplementary information). The catalytic oxidation of AA and AD by IMWCNTGCE was also studied by chronoamperometry. Chronoamperograms are recorded at a potential step of 70 mV for AA and 200 mV for AD (Figs. S6 and S7). Using the Cottrell equation (Bard and Faulkner, 2001), a diffusion coefcient was calculated as 1.77 10 6 cm2 s 1 for AA. Also, the diffusion coefcient for AD was found as 8.26 10 6 cm2 s 1 (see S3, Supplementary information). 3.4. Differential pulse voltammetric measurements The main objective in this study was the development of a modied electrode capable of electrocatalytic oxidation of AA and AD and also simultaneous detection of AA, AD, AC, and Trp. Fig. S8A shows differential pulse voltammograms (DPVs) obtained for the oxidation of different concentrations of AA at the IMWCNTGCE surface. Also, Fig. S8BD shows that the plot of the peak current versus the AA concentration is constituted of three linear segments of 3.144.1 mM, 44.1555.5 mM, and 555.5 3571.4 mM with different slopes. Differential pulse voltammetry (DPV) was also used to determine the linear range and the detection limit of AD. Fig. S9A shows DPVs obtained for the oxidation of different concentrations of AD at IMWCNTGCE. The plot of peak current versus AD concentration (1.3833.3 mM) is shown in Fig. S9B. The calibration plot was linear for three concentration ranges, 1.38.8 mM, 8.879.4, and 79.4833.3 mM of AD (Fig. S9C E) with different slopes. According to the method

mentioned by Bard and Faulkner (2001), the lower detection limit, Cm, was obtained by using the equation Cm 3sb/m, where sb is the standard deviation of the blank response (mA) and m is the slope of the calibration plot. The data analysis presents the lower limits of detection for AA and AD as 0.96 mM and 0.38 mM, respectively. The average voltammetric peak current and the precision for the repeated measurements (n 7) of 15.0 mM AA and 5.0 mM AD at IMWCNTGCE were 3.92 7 0.10 and 0.72 7 0.02 mA, respectively. Tables S1 and S2 show some of the response characteristics obtained for AA and AD in this study in comparison to those previously reported by others. These data show that the responses of the proposed modied electrode are superior in most cases to those of the previously reported modied electrodes. Based on these results, the bifunctional sensor is able to determine four compounds simultaneously, while in most of the other works, it has been reported for the measurement of two or three compounds. The next attempt was made to detect AA, AD, AC, and Trp simultaneously by using an IMWCNTGCE. Fig. 4A shows the DPVs obtained for the oxidation of different concentrations of AA, AD, AC, and Trp at the IMWCNTGCE surface. Therein, one can see four well distinguished anodic peaks at potentials of 45 mV, 139 mV, 331 mV, and 655 mV corresponding to the oxidation of AA, AD, AC, and Trp, respectively. The calibration curves for AA, AD, AC, and Trp were linear for the concentration ranges of 681.82500.0 mM of AA, 113.6416.7 mM of AD, 136.4500.0 mM of AC, and 227.3833.3 mM of Trp (Figs. 4B E). The correction corresponding to the background current was considered for all the measured currents in this study according the method that described in Bard and Faulkner (2001). As it can be seen, the sensitivity of the modied electrode for AA and AD detection in the presence and absence of another tree analytes is almost the same. Therefore, it is possible to individually or simultaneously determine AA, AD, AC, and Trp in mixture samples at IMWCNTGCE without any cross interferences. The inset of Fig. 4A shows the DPV of a mixture of 2000.0 mM AA, 350.0 mM AD, 450.0 mM AC, and 800.0 mM Trp at a bare GCE; the bare GCE could not separate the voltammetric signals of AA, AD, AC, and

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Trp. However, in the case of IMWCNTGCE, four anodic peaks corresponding to the oxidation of AA, AD, AC, and Trp were obtained. The utility of the proposed modied electrode for determining AA, AD, and AC in pharmaceutical formulations was tested by a differential pulse voltammetric method. The modied electrode was used to determine AA concentration in an injection solution and tablets as real samples, AD concentration in an adrenaline injection solution (1 mg mL 1), and AC concentration in an oral solution and tablet samples. The AA and AD injection solutions and the AC oral solution were diluted 755, 34, and 794 times, respectively. 100 mL of double distilled water with one AA tablet (4.015 g) dissolved in it was diluted 35 times with a 0.15 M phosphate buffer solution before the measurements. Also, for the measurement of AC in a tablet sample, 500 mL of double distilled water with one AC tablet (0.417 g) dissolved in it was diluted 21 times with a phosphate buffer solution. Then, the diluted sample solutions were placed in an electrochemical cell to determine their concentrations using the DPV method. The obtained results are listed in Table 2. To verify the validity of the results, the samples were spiked with certain amounts of AA, AD, AC, and Trp at levels similar to those of the samples themselves. The results in Table 2 show that RSD% and recovery rates of the spiked samples were acceptable. The reliability of the proposed modied electrode was also evaluated by comparing the obtained results with those declared on the labels of the pharmaceutical products. Based on the currents of repeated DPVs (n 3) and using the calibration plots which are shown in Figs. 4B D, the AA concentration in injection solution of AA and tablet of AA, AD concentration in injection solution of AD, and AC concentration in an oral solution and tablet sample were obtained as 98.8 7 2.6 mg mL 1, 126.1 7 3.7 mg g 1, 0.98 7 0.22 mg mL 1, 23.5 7 0.67 mg mL 1 and 771.6 7 17.4 mg g 1, respectively. The data in Table S3 demonstrate that the results obtained by a differential pulse voltammetric method are in very good agreement with the values declared on the labels of the samples. This suggests that the detection procedures have been free from any interference on the part of the sample matrix.

bifunctional electrocatalyst for oxidation of AA and AD. In DPV measurements, there appeared three linear calibration ranges for AA and AD. The modied electrode not only improved the electrochemical catalytic oxidation of AA and AD but also was used for simultaneous determination of AA, AD, AC, and Trp in mixture samples. This study has also demonstrated the practical analytical utility of the modied electrode for the determination of AA, AD and AC in a pharmaceutical product.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2012.09.028.

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Conclusions In the present study, 4-(1H-benzo[d]imidazol-2-ylthio)-5methylbenze-1,2-diol, as an imidazole derivative, was synthesized and applied as an electron transfer mediator. The imidazole electrodeposited at a multi-wall carbon nanotubes modied glassy carbon electrode surface (IMWCNTGCE) shows two pairs of redox couple in a phosphate buffer solution (pH 7.0). The results show that the redox couples play the role of a