Ni(OH)2 nanoparticles-carbon nanotube composite modified glassy carbon electrode as a sensor for simultaneous determination of dopamine and serotonin in the presence of ascorbic acid

Sensors and Actuators B 176 (2013) 543–551

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

Nafion/Ni(OH)2 nanoparticles-carbon nanotube composite modified glassy carbon electrode as a sensor for simultaneous determination of dopamine and serotonin in the presence of ascorbic acid Ali Babaei a,b,∗ , Ali Reza Taheri a a b

Department of Chemistry, Arak University, Arak, 38156-8-8349, Iran Research Center for Nanotechnology, Arak University, Arak, 38156-8-8349, Iran

a r t i c l e

i n f o

Article history: Received 12 May 2012 Received in revised form 6 September 2012 Accepted 9 September 2012 Available online 16 September 2012 Keywords: Dopamine Serotonin, Nafion Ni(OH)2 nanoparticles Multiwalled carbon nanotubes Modified glassy carbon

a b s t r a c t The electrochemical oxidation of dopamine (DA) and serotonin (ST) have been investigated by application of Nafion/Ni(OH)2 -multiwalled carbon nanotubes modified glassy carbon electrode (Nafion/Ni(OH)2 -MWNTs/GCE) using cyclic voltammetry (CV), differential pulse voltammetry (DPV) and chronoamperometry (CA) methods. The modified electrode worked as an efficient electron-mediator for DA and ST in the presence of ascorbic acid (AA). Voltammetric techniques separated the anodic peaks of DA and ST, and the interference from AA was effectively excluded from DA and ST determination. The DPV data showed that the obtained anodic peak currents were linearly proportional to concentration in the range of 0.05–25 ␮mol L−1 with a detection limit (S/N = 3.0) of 0.015 ␮mol L−1 for DA and in the range of 0.008–10 ␮mol L−1 and with a detection limit of 0.003 ␮mol L−1 for ST. The proposed sensor was used for determination of ST and DA in human blood serum with satisfactory results. © 2012 Elsevier B.V. All rights reserved.

1. Introduction There is considerable interest in developing electrochemical techniques for determination of neurotransmitters such as dopamine (DA) and serotonin (ST). DA is a ubiquitous neurotransmitter (NTM) in mammalian brain tissues that plays an important physiological role in the functioning of central nervous, renal, hormonal and cardiovascular systems as an extra cellular chemical messenger [1,2]. DA belongs to the family of inhibitory neurotransmitters; its function is to regulate neural interactions by reducing the permeability of gap junctions between adjacent neurons of the same type. Serotonin (5-hydroxytryptamine or ST) is a monoamine neurotransmitter synthesized in serotonergic neurons in the central nervous system and plays a crucial role in the emotional system together with other monoamine transmitters such as regulation of mood, sleep, emesis (vomiting), sexuality and appetite. Low levels of ST have been associated with several disorders, notably depression, migraine, bipolar disorder and anxiety [3,4]. In addition, neurodegeneration of ST- and DA-containing neurons contributes to late-onset neurological diseases, including Parkinson’s and

∗ Corresponding author at: Arak University, Department of Chemistry, Arak 38156-8-8349, Iran. Tel.: +98 861 4173401; fax: +98 861 4173406. E-mail addresses: [email protected], [email protected] (A. Babaei). 0925-4005/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2012.09.021

Alzheimer’s diseases, and possibly to normal ageing of the brain [5]. Besides numerous reports have shown their coexistence in biological systems and that they influence each other in their respective activities [6,7]. Therefore investigation of neurological behavior and also simultaneous determination of DA and ST is of great importance for the elucidation of their precise physiological functions. A range of analytical techniques such as chromatographic methods [8], mass spectroscopy [9], spectrophotometry [10] and chemiluminesence [11] are reported in the literature for detection of DA and ST. However these methods suffer from some disadvantages including long analysis times, high costs, the requirement for sample pretreatment, and in some cases low sensitivity and selectivity. These disadvantages probably make them unsuitable for routine analysis. The advantages of electrochemical methods for determination of DA and ST include low cost, high sensitivity and short measurement time [4,12–14]. However, the electrochemical measurement of neurotransmitter concentrations has been mainly unsatisfactory due to the inability of the electrodes employed to separate the potentials of these species sufficiently to allow for accurate detection. AA is usually present in vivo at concentrations 100–1000 times higher than the NTMs. Obviously, it is necessary to develop selective and sensitive techniques to resolve these problems. Therefore, various electrochemical approaches have been made to overcome these difficulties for the determination of DA and ST [3,4,12,15–17].

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To prevent loss of the materials from the electrode surface and to improve the anti-interferential ability of the sensor, Nafion films have been used extensively for the construction of biosensors. Nafion, a perfluorosulfonated derivative of Teflon, is a cationexchange polymer whose films are highly permeable to cations but almost impermeable to anions [18–21]. In the pH range of 5.27–8.87, ascorbic acid (AA) (pKa = 4.10) and uric acid (UA) (pKa = 5.27) exist in anionic form, while DA (pKb = 8.87) and ST (pKb = 7.59) are in cationic form. Thus, under physiological conditions a Nafion membrane strongly repulses anionic AA and UA and highly attracts cationic DA and ST and it has been successfully used in selective determinations of DA [22,23]. Nafion, with its good electrical conductivity, high chemical stability and good biocompatibility, has been widely used as an easily fabricated protective electrode coating material. An additional advantage with the use of Nafion is the improvement in terms of robustness towards mechanical damage [24]. Nagy et al. [18] demonstrated that a graphite electrode coated with Nafion film could be used to determine DA, norepinephrine and 5-hydroxytryptamine. Rocha et al. [25] reported that a negatively-charged Nafion film on a glassy carbon electrode could respond selectively to DA oxidation and eliminate the interference of AA. The discovery of carbon nanotubes (CNTs) has attracted much attention due to their structural uniqueness, chemical and physical properties and potential applications [26,27]. The subtle electronic behavior of CNTs reveals that they have the ability to mediate electron transfer reactions of electroactive species in solution when used as an electrode modifier. Additionally, transition-metal nanoparticles, in different forms, have emerged as a novel family of catalysts able to promote more efficiently a variety of organic transformations because of their small size and extremely large surface-to-volume ratio [28,29]. Recently several different types of nanoparticles have been successfully introduced onto CNTs, such as CdTe [30], Au [31], Cu [32], Ag [33] and Ni(OH)2 [34] for fabrication of sensors. Ni, NiO, Ni(OH)2 particles and nanoparticles have also been used to modify traditional electrode surfaces such as diamond [35], gold [36], carbon or graphite [37,38]. In contrast to Ni nanomaterials which are unstable and easily oxidized in air and solution, the hydroxide and oxide of nickel (II) are relatively stable [38,39]. Nano nickel hydroxide with a small crystalline size shows a high proton diffusion coefficient, which leads to excellent electrochemical performance. Many methods for preparing nano scale nickel hydroxide such as coordination precipitation, precipitation transformation, hydrothermal conversion, urea homogeneous precipitation, and micro emulsion, need harsh reaction conditions such as high temperature or pressure [40]. Some methods use organic solvents which increase the production cost [41]. Other methods require the addition of an excess of precipitating agent to the reaction system resulting in impure product [38,39,42–45]. However the method of coordination homogeneous precipitation (CHP) is new and facile, without needing expensive raw materials or equipment. It is also easy for mass production, and can be applied to the synthesis of various hydroxide or oxide nanocrystals [42,45,46]. In this work, we developed a composite coated glassy carbon electrode (GCE), which contains multiwalled carbon nanotubes (MWNTs), Ni(OH)2 nanoparticles (fabricated by CHP method) and Nafion, based on the idea that the MWNTs with Ni(OH)2 nanoparticles could facilitate the electron transfer, due to the synergistic electrocatalysis effects. Nafion is used because of its immobilization matrix capability and cation-exchange properties. The fabricated Nafion/Ni(OH)2 /MWNTs/GCE sensor results in excellent sensitivity, wide linear range, favorable stability and reproducibility and a low detection limit. The analytical performance of the sensor has been evaluated in human blood serum with satisfactory results.

2. Experimental 2.1. Reagents and solutions DA and ST were obtained from Acros and Sigma chemical companies, respectively. Nafion, a 5 wt.% solution in a mixture of lower aliphatic alcohols and 10% water, was obtained from Aldrich. MWNTs (>95 wt.%, 5–20 nm) was purchased from PlasmaChem GmbH company. 0.1 M phosphate buffer solution (PBS) was prepared by dissolving appropriate amounts of sodium hydrogen phosphate and sodium dihydrogen phosphate in a 250 mL volumetric flask. The pH of the buffer was adjusted to appropriate value by addition of 7.5 M sodium hydroxide solution. All electrochemical experiments were carried out in 0.1 M PBS at pH 7.0. Freshly prepared ST, DA, UA and AA solutions were used for each experiment. Other reagents were of analytical grade purchased from Merck and used without further purification. All aqueous solutions were made with triply distilled water. Fresh human serum samples were available from Razi institute of vaccine and serum company (Tehran, Iran). 2.2. Synthesis of nanoscale Ni(OH)2 Nanoscale Ni(OH)2 was synthesized using a simple coordination precipitation procedure as previously reported [42]. Briefly, by adding concentrated ammonia (28 wt.%) to nickel (II) nitrate solution (1 M), a deep blue colored nickel hexammine complex solution was formed and added into a given amount of distilled water, the reaction was carried out under magnetic stirring for 1 h at 70 ◦ C. Finally, light green sediments were formed. The precipitate was separated by centrifuge and rinsed with distilled water and ethanol three times respectively to remove the adsorbed ions, then dried in a vacuum oven at 80 ◦ C for 12 h. The final product was a green powder. Product that is obtained without any surfactant in the reaction process was platelet-like shape. 2.3. Electrode modification Prior to use, the glassy carbon electrode (GCE, 2 mm in diameter) was first polished with alumina slurry (1.0 ␮m followed by 0.05 ␮m) and ultrasonically cleaned with 1:1 water, ethanol and distillated water. A stock solution of MWCNTs/Ni(OH)2 in DMF was prepared by dispersing weighed amounts of MWNTs and Ni(OH)2 nanoscale (95%:5%, w/w) in 1 mL DMF using an ultrasonic bath until a homogeneous suspension resulted, and 20 ␮L of prepared suspension was casted on the electrode with a microsyringe. After that, Nafion/Ni(OH)2 /MWCNT/GCE was prepared by coating the electrode surface with 8 ␮L of 5.0% (v/v) Nafion solution diluted with ethanol. During these procedures a small bottle was fitted tightly over the electrode so that the solvent could evaporate slowly and a uniform film was formed (see Fig. S1 in Supporting information). The fabricated electrode was stored at 4 ◦ C when not in use. For comparison, Nafion/GCE, Nafion/Ni(OH)2 /GCE and Nafion/MWNTs/GCE were prepared and used for further investigation. 2.4. Instrumentation All the voltammetric measurements were carried out using our Nafion/Ni(OH)2 nanoparticles/carbon nanotube composite modified glassy carbon electrode, (Nafion/Ni(OH)2 /MWCNT/GCE) as the working electrode, Ag/AgCl 3 mol L−1 KCl as the reference electrode and platinum wire as an auxiliary electrode. Differential pulse voltammetry (DPV), cyclic voltammetry (CV) and chronoamperometry (CA) experiments were carried out in nitrogen-saturated

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Fig. 1. SEM image of the MWNTs and Ni(OH)2 nanoscale on glassy carbon (a) and TEM image of nickel hydroxide powders (b).

water by using an Autolab PGSTAT 30 Potentiostat Galvanostat (EcoChemie, The Netherlands) coupled with a 663 VA stand (Metrohm, Switzerland). All potentials given are with respect to the potential of the reference electrode. The pH measurements were performed with a Metrohm 744 pH meter using a combination glass electrode. X-ray diffraction (XRD) measurements were performed at a speed of 0.01◦ s−1 by a Bruker Axs diffractometer (Germany) with Cu K␣ ( = 1.5418 nm) operating at 40 kV, 30 mA. The morphology of the nano scale Ni(OH)2 was investigated by scanning electron microscopy (SEM, Leica Cambridge, model S 360) and transmission electron microscopy (TEM, Philips CM10). 2.5. General procedure Each sample solution (10 mL) containing 0.1 mol L−1 phosphate buffer solution (pH 7.0) and an appropriate amount of DA and ST was pipetted into a voltammetric cell. Unless stated otherwise, all voltammetry measurements were preceded by 90 s accumulation time at open circuit potential in analyte solution. The voltammograms were recorded by applying positive-going potential from −0.1 to 0.6 V. The voltammograms showed anodic peaks around 0.15 and 0.35 V corresponding to the DA and ST compounds with their currents being proportional to their concentrations in the solutions. Calibration curves were obtained by plotting the anodic peak currents of DA and ST against the corresponding concentrations. The electrode was regenerated by successive washing with triply distilled water and then 0.5% sodium hydroxide solution. The electrode was finally rinsed carefully with distilled water to remove all adsorbate from the electrode surface and to provide a fresh surface before running subsequent experiments.

The XRD pattern of the nanoscale Ni(OH)2 is exhibited in Fig. 2. It can be seen that several diffraction peaks appear at 2 = 19◦ , 34◦ , 39◦ , 52◦ , 58◦ , 62◦ , 71◦ and 74◦ , which can be indexed to planes (0 0 1), (1 0 0), (1 0 2), (1 1 0), (1 0 2), (1 1 1), (1 0 3) and (2 0 1) of ␤Ni(OH)2 according to JCPDS card no. 14-0117, respectively. Previous reports have indicated that peaks (0 0 1) and (1 0 l) were especially broad when the nickel hydroxide was more active [47–49]. Delmas and Tessier [48] also considered a correlation between the electrochemical activity and the XRD pattern of nickel hydroxide. Also, another work from Delmas and co-workers [50] determined this type of pattern to be associated with very poorly crystallized nickel hydroxide, denoted as ␤bc (bc: badly crystallized). This material, obtained by the ageing of ␤-nickel hydroxide, has very broad (0 0 1) and (1 0 1) lines in its XRD pattern, with narrow (h k 0) lines. However the XRD pattern of this sample is very similar to the ␤bc-nickel hydroxide. A broadening of the (1 0 1) line for nanoscale Ni(OH)2 can be seen, which is ascribed to the disordered structure of the material. Thus, the broadening of the (0 0 1) reflection is caused by the smaller crystalline size, as previously reported [51]. 3.2. Electrochemical behavior of Nafion/Ni(OH)2 /MWNTs/GCE The electrochemical behavior of DA and ST at the different composite films in pH 7.0 phosphate buffer was examined using CV. Fig. 3 shows the cyclic voltammograms of GCE, Nafion/GCE, Nafion/Ni(OH)2 /GCE, Nafion/MWNTs/GCE, Nafion/Ni(OH)2 /MWNTs/GCE in deoxygenated 0.1 mol L−1 phosphate buffer solution for DA (Fig. 3a) and ST (Fig. 3b). It can be seen (curves A in Fig. 3) that a broad oxidation peak and ill-defined redox waves appeared at the bare electrode and the peak potentials of DA and ST were indistinguishable. The Nafion-modified GCE significantly enhanced the redox peak currents compared with the bare GCE (curves B in Fig. 3). The Nafion film can enhance the surface

3.1. Characterization of Ni(OH)2 nanoparticles Nanoscale Ni(OH)2 was characterized by means of SEM and TEM. Fig. 1a shows a typical image of the nanoscale Ni(OH)2 synthesized via coordination precipitation method and MWNTs. It can be observed that it appears to have a platelet-like shape and with a dimension of 50–80 nm, and weak agglomeration can be seen. Fig. 1b displays TEM image of Nanoscale Ni(OH)2 . The result shows the nanoparticles are in the same sizes as it is shown in the SEM image.

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3. Result and discussion

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Fig. 3. CVs recorded of 10 ␮mol/L DA (a) and ST (b) at GCE (A), Nafion/GCE (B), Nafion/Ni(OH)2 /GCE (C), Nafion/MWCNT/GCE (D) and Nafion/Ni(OH)2 /MWCNT/GCE (E) Scan rate: 100 mV/s.

concentration of DA and ST via ion-exchange and consequently improve selectivity at the Nafion film coated electrode. It can also be seen that the peak currents of DA and ST increases further at the Ni(OH)2 /Nafion modified GCE as a result of the nano-size effect of Ni(OH)2 nanoparticles (curves C in Fig. 3).The enhancement in peak currents and the lowering of overpotentials are clear evidences of the catalytic effects of Ni(OH)2 toward the DA and ST redox reaction. Curves D in Fig. 3 demonstrate that Nafion/MWNTs can effectively catalyze the electrooxidation of DA and ST and greatly improve the peak shapes. This can mainly be attributed to the large surface area, subtle electronic properties of MWNTs and the ion-exchange characters of Nafion; meanwhile, the oxidation peak potential (Epa ) shifts negatively, and the reduction peak potential (Epc ) shifts positively. The modified Nafion/MWNTs/GCE not only improves the redox peak currents but also makes the redox reaction of DA more reversible. Curves E in Fig. 3 strongly suggests that the hybrid film of Nafion, MWNTs and Ni(OH)2 can combine the advantages of all of them and accelerate electron transfer significantly; therefore, result in remarkably increased response towards the redox of DA and ST in contrast to Nafion or MWNTs/Ni(OH)2 alone. 3.3. Optimization of experimental variables Modification of a GCE with different amounts of Nafion, MWCNTs and Ni(OH)2 nanoscale was tested (n = 5). The amount of nanoscale Ni(OH)2 influences the sensitivity of the sensor. It was found that as the composition of nanoscale Ni(OH)2 increased from 2 to 5%, the response of the electrode improved and when

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the composition was more than 5%, the response decreased with a larger background current, which resulted in poor measurements for DA and ST (Fig. 4A). So 20 ␮L of 5% nanoscale Ni(OH)2 (95% MWNTs) was chosen for the fabrication of the sensor. The influence of the amount of Nafion on the analytical characteristics of the modified electrode is a vital factor affecting the analytical sensitivity of the biosensor. At optimum pH of 7.0, the Nafion membrane electrostatically attracts DA and ST and rejects AA and UA because DA and ST are in their cationic forms while AA and UA exist in their anionic forms. But on the other hand Nafion has weak electrical conductivity and too much Nafion would decrease the mass transfer rate. Therefore, the amount of Nafion will affect the performance of the modified electrode. Fig. 4B displays the effect of the amount of Nafion in the modified electrode. The largest anodic current was achieved when 8.0 ␮L of Nafion 5.0% (v/v) solutions was used, and it would decrease for increasing volumes of Nafion. To learn more about the adsorption of DA and ST at the Nafion/Ni(OH)2 /MWNTs film-coated GCE, we examined the influences of accumulation potential and time. The oxidation peak currents of DA and ST after 2 min accumulation at different potentials from 0.50 V to 0.10 V remained almost stable, revealing that potential does not affect the oxidation peak currents of DA and ST. In contrast to potential, accumulation time strongly influences the peak currents. Fig. 5c shows the variations of differential pulse anodic peak currents of 10 ␮mol L−1 DA and ST with respect to accumulation times. The anodic peak currents of DA and ST improve with accumulation time, but after 90 s for DA and after 60 s for ST remained almost stable. This may be due to saturation of the amount of DA and ST adsorbed on the modified electrode surface.

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Fig. 4. Effect of the ratio of Ni(OH)2 nano scale on the sensor response. (a)The influence of the amounts of Nafion on the response of the sensor, (b) the variations of differential pulse anodic peak currents of 10 ␮mol/L DA and ST with respect to accumulation time (c) (n = 5). Pulse amplitude: 50 mV; pulse width: 50 ms; scan rate = 20 mV/s.

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Fig. 5. Cyclic voltammograms of Nafion/Ni(OH)2 /MwNTs/GCE in 0.1 M PBs at different pHs from 4 to 8 in the presence of 10 ␮M of DA and ST (a), plot of peak current vs. pH values (b), plots of E vs. pH (c), scan rate: 100 mV s−1 .

Thus, the accumulation time of 90 s was selected as an optimum time for subsequent experiments.

on the surface of Nafion/Ni(OH)2 /MWNTs in the studied range of potential sweep rates, with following equations:

3.4. Effect of pH

DA : ipa = 0.124n + 7.67 (R2 = 0.998)

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The effect of pH of supporting electrolyte on anodic peak currents was shown in Fig. 5a to obtain the best anodic peak resolution (Epa ) and maximum sensitivity in mixture solutions of DA and ST. In these investigations, cyclic voltammetric studies were performed in various buffered solutions in the pH 4.0–9.0 range for mixture solutions of DA and ST. Variation of the corresponding oxidative potential (Epa ) with pH obeyed the following equations:

ST : ipa = 0.130n + 8.23 (R2 = 0.999)

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DA : Epa (V) = −0.058pH + 0.555 (R2 = 0.994)

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ST : Epa (V) = −0.052pH + 0.711 (R2 = 0.997)

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The slope of the variation of Epa as a function of solution pH is close to the Nernstian slope of 0.059 V/pH unit at 25 ◦ C, which indicates an equal number of electrons and protons involved in the electrochemical oxidations of DA and ST. As shown in Fig. 5b and c, the best peak separation can be seen at pH 6.0. However in solution with pH 7.0, higher anodic peak currents for both drugs are resulted and reasonable peak separation was achieved. Fig. 5c shows that oxidation potentials of DA and ST shift to less positive potential with increasing solution pH. This is a consequence of the oxidation process having a deprotonation that is facilitated at higher pH values and suggesting the production of two electrons and two protons per molecule of DA and ST oxidized, which is in agreement with literature reports [13,16,52,53]. 3.5. Effect of the scan rate The effect of scan rate on the oxidative potential (Epa ) and peak current (Ipa ) of DA and ST (Fig. 6) at the surface of Nafion/Ni(OH)2 /MWNTs/GCE was studied and the cyclic voltammetric curves of DA and ST obtained in the range 0.01–0.4 V s−1 in order to investigate the kinetics of electrode reactions. The results showed that the peak currents vary linearly with the scan rate (insets of Fig. 6) for both drugs, which confirms an adsorptioncontrolled process for electrochemical oxidation of DA and ST

The Epa shifted to more positive values with increasing the scan rate (), suggesting the electron transfer is quasi-reversible. The ˛n␣ value (˛, the electron transfer coefficient; n␣ , number of electrons involved in the oxidation) of ST can be evaluated as 0.983 based on the slope of the Epa versus log  plot. If ˛ is assumed to be 0.5 [54], the value of n␣ is estimated as 1.96, indicating that two electrons were involved in the oxidation of ST [17].

3.6. Voltammetric responses of DA and ST To learn more about the electrochemical responses when DA and ST coexist, we tested the voltammetric behaviors of DA (or ST) in the presence of a large excess of AA by DPV in pH 7.0 phosphate buffer (Fig. 7). The oxidation peak currents of DA or ST increased with prolonging preconcentration time and finally reached a plateau. The electrochemical response of additions of DA from 0.05 to 25 ␮mol L−1 in the co-existence of 3 ␮ mol L−1 ST and 0.5 mM AA under the optimized conditions by DPV techniques using Nafion/Ni(OH)2 /MWNTs/GCE is depicted in Fig. 7a. With application of the DPV method two linear ranges were obtained. The first linear dynamic range was from 0.05 ␮M to 1 ␮M, with a calibration equation of Ip (␮A) = 1.991c (␮M) + 0.005 (R2 = 0.9992) and the second linear dynamic range was 1–25 ␮M with a calibration equation of Ip (␮A) = 2.4c (␮M) + 0.8 (R2 = 0.9990). A detection limit of 0.0151 ␮M (S/N = 3) was obtained. Fig. 7b shows differential pulse voltammograms and corresponding calibration curves obtained from 0.008 to 10 ␮mol L−1 of ST with coexisting of 3 ␮mol L−1 DA and 0.5 mM AA. The first linear dynamic range was from 0.008 to 0.2 ␮M, with a calibration equation of Ip (␮A) = 4.991c (␮M) + 0.018 (R2 = 0.9991) and the second linear dynamic range was between 0.2 and 10 ␮M with a calibration equation of Ip (␮A) = 2.596c (␮M) + 0.01 (R2 = 0.9988). A detection limit of 0.003 ␮M (S/N = 3) was obtained.

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Fig. 6. Cyclic voltammograms of (a) ST, (b) DA at different scan rates (from A to L) 10, 25, 75, 100, 125, 150, 200, 250, 300, 350 and 400 mV s−1 . (Insets) dependence of peak currents vs. scan rate.

To evaluate the repeatability of the Nafion/Ni(OH)2 /MWNTs/ GCE, the peak currents of 20 successive measurements by DPV in a mixture solution of 10 ␮M DA, 10 ␮M ST were determined. The relative standard deviation (R.S.D.) of 2.2% and 2.2% was obtained for DA and ST, respectively, indicating that the Nafion/Ni(OH)2 /MWNTs/GCE is not subject to surface fouling by the oxidation products. The stability of the proposed sensor was investigated. After 100 cyclic runs, the voltammetric response to 10 ␮M DA and 10 ␮M ST almost remained 93% and 94% of the initial response, respectively (data not shown). The storage stability of the proposed biosensor was also studied. When not in use, the electrode was suspended above PBS at 4 ◦ C in a refrigerator. The response to 10 ␮M DA was tested intermittently. After 7 and 15 days at 4 ◦ C, the sensor retained 92% and 90% of its initial response current, respectively. In addition the response to 10 ␮M ST was also tested and the sensor retained 91% and 86% of its initial response current after 7 and 15 days, respectively. Indicating that the Nafion/Ni(OH)2 /MWNTs film has a long lifetime.

nearly no interference for the determination of DA and ST, suggesting that the Nafion/Ni(OH)2 /MWNTs modified GCE responds selectively to DA and ST. At pH 7.0, AA and UA exist in their anionic form, but DA and ST are cationic. Nafion on the GCE surface selectively attracts cationic species and allows them to pass through to the electrode surface. In contrast, anionic species were prevented from reaching the electrode surface. Unlike DA and ST, AA and UA did not exchange electrons with the electrode owing to the selective permeation of the Nafion film. Interference studies were also performed with other compounds. The tolerance limit was defined as the maximum concentration of the interfering substance that causes an error less than 5% for determination of DA and ST. It was found that a 200-fold excess of 3,4-dihydroxyphenylacetic acid (DOPAC, known as the metabolite of DA), 300-fold excess citric acid, 150-fold excess glutamic acid, 100-fold excess of glucose, 400-fold excess NaCl, 500-fold excess KCl, 450-fold excess MgCl2 , 400-fold excess CaCl2 , 500-fold excess Ca(NO3 )2 and 220-fold excess of oxalate did not interfere with the measurement of 10 ␮M DA and ST. It shows that the proposed method is free from interferences of the most common interfering agents.

3.8. Effects of interferences on the behaviors of DA and ST

3.9. Determination of DA and ST in human blood serum

Voltammograms of DA and ST in the presence of AA were investigated (see Fig. S2 in Supporting information). Upon addition of 10 ␮mol L−1 DA and ST oxidation peaks appeared with good sensitivity and even 800 and 500 times AA and UA respectively had

The Nafion/Ni(OH)2 /MWNTs/GCE was next utilized for the simultaneous determination of DA and ST in human blood serum. Abnormal concentrations of ST and DA in serum have been shown to reflect the derailed serotonergic and dopaminergic function

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Fig. 7. DPVs of DA at Nafion/Ni(OH)2 -MWNTs/GCE in the presence of 3 ␮mol L−1 ST and 0.5 mM AA in pH 7.0 PBS. DA concentration (from A to L): 0.05, 0.15, 0.5, 1, 2, 3, 5, 7, 10, 15, 20, 25 ␮mol L−1 (a). DPVs of ST at Nafion/Ni(OH)2 -MWNTs/GCE in the presence of 3 ␮mol L−1 DA and 0.5 mM AA in pH 7.0 PBS. ST concentration (from A to L): 0.008, 0.015, 0.03, 0.05, 0.1, 0.2, 0.5, 1, 1.5, 2.5, 5, 10 ␮mol L−1 (b). (Insets) The calibration curves of related species.

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Table 1 Determination of DA and ST in diluted human serum with Nafion/Ni(OH)2 -MWNTs/GCE. Analyte

Added (␮M)

proposed methoda (␮M)

Recovery (%)

Standard method (␮M)

ST

0.00 0.20 0.40 0.60 0.80 1.00 2.00

0.48 ± 0.02 0.67 ± 0.05 0.85 ± 0.04 1.07 ± 0.06 1.29 ± 0.06 1.44 ± 0.07 2.41 ± 0.05

– 98.53 96.59 99.07 100.78 97.30 97.18

0.51 ± 0.04 – – – – – –

DA

0.00 0.20 0.40 0.60 0.80 1.00 2.00

– 0.19 ± 0.04 0.38 ± 0.05 0.61 ± 0.07 0.78 ± 0.06 0.98 ± 0.07 1.97 ± 0.09

– 95.00 95.00 101.67 97.50 98.00 98.50

– – – – – – –

a

Average of ten determinations at optimum conditions.

Table 2 Comparison of the proposed electrode for DA and ST with other types of nanocomposite material modified electrode. Electrode

Method

LDR (␮M)

DL (␮M)

References

DNA-PPyox/CFE

DPV

DA ST

0.3–10 0.01–1

0.05 0.007

[16]

MWNT-DHP/GCE

DPV

DA ST

0.05–5 0.02–5

0.011 0.005

[12]

MWNT-IE

DPV

DA ST

0.5–10 1–15

0.1 0.2

[4]

EPPGE

DPV

DA ST

0.2–25 0.1–100

0.09 0.06

[13]

PPS/GC

DPV

DA ST

50–500 50–500

20 20

[15]

ACh/GCE

DPV

DA ST

0.7–5 1–30

0.3 0.5

[51]

Nano-Au/PPyox/GCE

DPV

DA ST

0.75–20 0.07–2.2

0.15 0.001

[56]

5-HTP/GCE

DPV

DA ST

0.5–35 5–35

0.31 1.7

[52]

Nafion/Ni(OH)2 /MWNTs/GCE

DPV

DA ST DA ST

0.05–25 0.008–10 0.25–22.78 0.15–14.80

0.0151 0.003 0.11 0.083

This work

Chronoamperometery

PPyox/CFE: overoxidized polypyrrole/carbon fiber electrode, DHP: dihexadecyl hydrogen phosphate, IE: intercalated graphite electrodes, EPPGE: edge plane pyrolytic graphite electrode, PPS: poly(phenosafranine), Ach: acetylcholine, 5-HTTP: 5-hydroxytryptophan.

in the central nervous system. Accurate determination of serum levels of ST and DA is necessary and useful. As a preliminary application in clinical studies, the modified electrode was used to assess the ST and DA concentrations in healthy human blood samples. The standard addition method was used to complete the experiment. When spiked with exogenous ST and DA (2 × 10−7 , 4 × 10−7 , 6 × 10−7 , 8 × 10−7 , 1 × 10−6 and 2 × 10−6 M), we obtained linearity with correlation coefficients of 0.9980 and 0.9984 for ST and DA, respectively. The measured serotonin values in blood samples from 10 different individuals were (4.8 ± 0.2) × 10−7 M which was in the normal range of 101–283 ng mL−1 (4.76 × 10−7 M − 1.33 × 10−6 M) according to the medical encyclopedia. DA was not detected in healthy blood. The typical DPV response of ST and DA with and without the spike of standard ST and DA solution is summarized in Table 1. The results by the Nafion/Ni(OH)2 /MWNTs/GCE for serotonin were compared with data obtained from analysis of the same plasma samples by the LC-EC assay method of Tagari et al. [55]. There is a good accordance between the proposed electrochemical data and the standard method.

4. Conclusions Simultaneous trace determination of DA and ST in the presence of AA was achieved by using Nafion/Ni(OH)2 /MWNTs/GCE. Large peak separations between DA and ST allow the detection and determination of DA and ST simultaneously at the Nafion/Ni(OH)2 /MWNTs/GCE using cyclic and differential pulse voltammetry. The modified GCE was fabricated by a layer-by-layer casting method, and the resulting electrode exhibited a good electrocatalytic performance to DA and ST because of the combining of Ni(OH)2 and MWCNTs. The electrodes were coated with Nafion, a perfluorosulfonated polymer which is practically impermeable to ascorbic acid and other anionic species and only slightly responsive to neutral metabolites. Thus it becomes selective for the cationic neurotransmitters, dopamine and serotonin. The electrode was used for simultaneous determination of DA and ST in real biological samples and satisfying results were achieved. The simple fabrication procedure, wide linear range, low detection limit, high stability and good reproducibility for repeated determination suggest that this electrode will be a good and attractive candidate for practical

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Biographies Ali Babaei received his BS degree in 1989 from Shahid Beheshti University, Tehran, Iran; MS degree in 1991 from Mazandarn University, Babolsar, Iran and PhD degree from Otago University, Dunedin, New Zealand. At present, he is associate professor of chemistry at Arak University, Arak, Iran. His main area of interest at present is electroanalytical chemistry. Ali Reza Taheri received his BS degree in 2003 from Isfahan University, Isfahan, Iran; MS degree in 2007 from Arak University, Arak, Iran. At present, he is a PhD student in chemistry department of Arak University, Arak, Iran.