Author’s Accepted Manuscript

Electrocatalytic oxidation and determination ofinsulin at nickel oxide nanoparticles-multiwalledcarbon nanotube modified screen printed electrode

Banafsheh Rafiee, Ali Reza Fakhari

PII: S0956-5663(13)00040-7DOI: http://dx.doi.org/10.1016/j.bios.2013.01.037Reference: BIOS5704

To appear in: Biosensors and Bioelectronics

Received date: 13 November 2012Revised date: 16 January 2013Accepted date: 21 January 2013

Cite this article as: Banafsheh Rafiee and Ali Reza Fakhari, Electrocatalytic oxidationand determination of insulin at nickel oxide nanoparticles-multiwalled carbon nanotubemodified screen printed electrode, Biosensors and Bioelectronics, http://dx.doi.org/10.1016/j.bios.2013.01.037

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Electrocatalytic Oxidation and Determination of Insulin at Nickel Oxide

Nanoparticles-Multiwalled Carbon Nanotube Modified Screen Printed

Electrode

Banafsheh Rafiee, Ali Reza Fakhari*

Department of Chemistry, Faculty of Sciences, Shahid Beheshti University, G. C., PO Box

19396-4716, Tehran, I. R. Iran

Abstract

Nickel oxide nanoparticles modified nafion-multiwalled carbon nanotubes screen printed

electrode (NiONPs/Nafion-MWCNTs/SPE) were prepared using pulsed electrodeposition of

NiONPs on the MWCNTs/SPE surface. The size, distribution and structure of the

NiONPs/Nafion-MWCNTs were characterized by transmission electron microscopy (TEM) and

x-ray diffraction (XRD) and also the results show that NiO nanoparticles were homogeneously

electrodeposited on the surfaces of MWCNTs. Also, the electrochemical behavior of

NiONPs/Nafion-MWCNTs composites in aqueous alkaline solutions of insulin was studied by

cyclic voltammetry, chronoamperometry and electrochemical impedance spectroscopy (EIS). It

was found that the prepared nanoparticles have excellent electrocatalytic activity towards insulin

oxidation due to special properties of NiO nanoparticles. Cyclic voltammetric studies showed

that the NiONPs/Nafion-MWCNTs film modified SPE, lowers the overpotentials and improves

electrochemical behavior of insulin oxidation, as compared to the bare SPE. Amperometry was

*Corresponding author, Tel.: +98 21 22431661; fax: +98 21 22431683. E-mail address: a-zavareh@sbu.ac.ir (A.R. Fakhari).

also used to evaluate the analytical performance of modified electrode in the quantitation of

insulin. Excellent analytical features, including high sensitivity (1.83 �A/μM), low detection

limit (6.1 nM) and satisfactory dynamic range (20.0 – 260.0 nM), were achieved under optimized

conditions. Moreover, these sensors show good repeatability and a high stability after a while or

successive potential cycling.

Keywords: Insulin; Nickel Oxide Nanoparticles; Amperometric Detection; Electrodeposition;

Screen Printed Electrode

1. Introduction

Insulin is a polypeptide hormone that contains one intra chain and two inter chain disulfide

bonds (supplementary material, S1). It serves as a predictor of diabetes of insulinoma and trauma

(Melloul et al., 2002; Tannuri et al., 1993) and it is used to control glucose levels in blood within

a narrow concentration range (Wang et al., 2007). The direct monitoring of insulin in a diabetic

patient has a better prospect in clinical investigation rather to the glucose measurement (Snider et

al., 2008; Kivlehan et al., 2008). Therefore, a fast and simple method for accurate detection of

insulin is very important.

Several methods have been explored for the quantitative analysis of insulin including enzyme-

linked immunosorbent assay (Kumada et al., 2007), radioimmunoassay (Murayama et al., 2006),

high performance liquid chromatography (HPLC) (Mercolini et al., 2008) and capillary

electrophoresis (Ortner et al., 2003). However, these methods are time-consuming, cumbersome,

and very costly. Direct electrochemical detection of insulin is simple and inexpensive and also is

attractive as it can provide sensitivity and reduces analysis time to enable continuous real-time

measurements compared to the above methodologies (Snider et al., 2008). However, direct

oxidation of insulin at conventional electrodes is limited by the slow kinetics and surface fouling

onto electrochemical devices (Zhang et al, 2005). Furthermore, low sensitivity, reproducibility

and stability over a wide range of solution compositions and high overpotential at electrodes are

other limitations of unmodified electrodes as an electrochemical sensor for insulin detection

(Salimi et al., 2008). Therefore, several electrochemical sensors (Wang et al., 2007; Zhang et al,

2005; Salimi et al., 2007; Salimi et al., 2008; Cox and Gray, 1989; Gorski et al., 1997; Pikulski

and Gorski, 2000; Cheng et al., 2001; Arvinte et al., 2010) were fabricated for trace and even

ultra-trace level detection of insulin.

Transition metals occur in biological systems because they have multiple oxidation states

separated by only modest potentials, which make them suitable candidates for electron-transfer

processes. Nickel is of particular interest as modifying agents because in basic media nickel

redox centers show high catalytic activity towards the oxidation of some organic compounds

(Casella et al., 1993; Ciszewski, 1995) at a relatively low cost, compared to other (especially,

noble metal) catalysts.

Carbon nanotubes (CNTs) have attracted much attention due to their unique properties. As

electrode modifiers, CNTs show negligible surface fouling (Pumera, 2009), decreased

overpotential, increased voltammetric currents, they can be used for promoting electron transfer

between the electroactive species and the electrode (Wang, 2005) and provide a new method for

fabricating electrochemical sensors or biosensors. It is well known that high-surface-area CNT

when combined with metal nanoparticles or metal oxides can improve the performance of the

final material (Arvinte, 2011). The antifouling properties of nickel oxide nanoparticles for

insulin, thiols, disulfidesand their oxidation products have been reported either (Salimi et al.,

2008). Recently, the preparation of a CNT-nickel-cobalt oxide modified graphite screen printed

electrode and its use in the electrocatalytic oxidation of insulin was reported by Arvinte et al.

(Arvinte et al., 2010). The modification was achieved by adsorption of NiCoO2 at a graphite

screen printed electrode.

In this work, we have reported a NiONPs/Nafion-MWCNTs modified screen printed electrode

which is prepared by the electrochemical pulsed potential deposition of the modifier. In fact,

special properties of nano NiO are used rather than NiO crystals as active sites on the MWCNTs.

The electrochemical behavior of NiONPs/Nafion-MWCNTs composites in aqueous alkaline

solutions of insulin was studied by cyclic voltammetry, chronoamperometry and electrochemical

impedance spectroscopy. Amperometry was also used for quantitative measurement of insulin.

2. Experimental

2.1. Chemicals and materials

Multiwalled carbon nanotubes with purity 95% (20 40 nm diameter and 1 10 μm length) were

obtained from Research Institute of Petroleum Industry (Tehran, Iran). Nafion 5% solution and

bovine insulin (�27 USP units/mg) were purchased from Sigma. All other solvents and reagents

were purchased from Aldrich or Merck and were used without further purification. A stock

solution of insulin (0.20 mM) was prepared daily by dissolving powdered insulin in water and

adding 10.0 μl of 1.0 M HCl to dissolve the powder. Insulin solutions were prepared by diluting

aliquots of the insulin stock solutions with a carrier solution. All solutions were prepared with

doubly distilled water. Insulin solutions were freshly prepared just prior to use and all

experiments were carried out at room temperature.

2.2. Instruments

All electrochemical experiments were performed by using Autolab potentiostat/galvanostat type

30 (2) (Eco Chemie, Netherlands), equipped with FRA and GPES 4.9 software. A screen-printed

carbon electrode (SPE) (3 mm in diameter) from Dropsens (Spain) was used as a planar three

electrode based on a graphite working electrode, a carbon counter electrode and a silver pseudo-

reference electrode. The electrode was rinsed in deionized water and preconditioned in 0.10 M

HCl solution by potential scanning in �0.4 to +1.0 V at a scan rate of 100 mV s-1. For the

amperometric measurements, the modified SPE was immersed in the stirred NaOH solution,

applying constant potential. EIS experiments carried out with a dc-offset potential of 550 mV

and in the frequency range of 100000 to 0.01 Hz. Transmission electron microscopy (TEM)

image was determined with a Philips CM10 TEM. A personal computer was used for data

storage and processing.

2.3. Preparation of modified electrode

MWCNTs were carboxylated (Ryu et al., 2009). The MWCNTs were washed with doubly

distilled water and dried in vacuum at 80 ºC. MWCNTs (10 mg) and 5.0 μl of 5.0% nafion

solution were dispersed in 5.0 ml water with ultrasonication for 1 h to get a homogenous

suspension. 10.0 μlof the suspension was casted onto the surface of screen printed electrode and

dried at room temperature.

The deposition bathes, were prepared using Ni(NO3)2.6H2O with a concentration of 40.0 mM.

The pH of the bath was adjusted in 2.0 using boric acid and some drops of sulfuric acid. The

pulse potential for making NiONPs on the electrode was applied using �0.4 V for 0.3 s and 0.0 V

for 2.5 s (supplementary material, S2). The sizes of particles were controlled by changing the

number of applied pulses in the depositions. Clearly the increasing of the pulses caused to the

formation of a larger particle of modifier at the surface of Nafion-MWCNTs/SPE. The prepared

electrode was conditioned in 0.10 M NaOH solution by potential cycling between 100 to 700 mV

for about 10 cycles of potential scans with a sweep rate of 100 mV s-1. These guidelines were

obtained experimentally as best values for complete transfhhh0ormation of Ni (��) to Ni (���) and

maximum activation of electrode surface towards electrocatalytic oxidation of insulin. All

electrochemical investigations were performed in the room temperature.

3. Results and discussion

3.1. Morphological characterization of the NiONPs/Nafion-MWCNTs/SPE

The surface morphology and elemental composition of the NiONPs/Nafion-MWCNTs/SPE was

characterized by TEM image and XRD, respectively (as shown in Fig. 1). The structure of the

composite showed a homogeneous film on the surface of SPE. Particles with diameter below

30.0 nm are observed in the TEM image, indicating a nanostructure for the NiO deposited film

on the Nafion-MWCNTs/SPE (Fig. 1; A). It is known that carboxylic acid groups on the surface

of MWCNTs can stimulate the deposition of NiO nanoparticles (Jin et al., 2007; Golovin et al.,

2011).

It is known that the physico-chemical properties of the NiO nanoparticles are very different from

bulk NiO crystals (Rao and Cheetham, 2006). An example of this is the high surface area and

excellent magnetic properties of ferromagnetic NiO nanocrystals which has attracted

considerable attention than the bulk NiO. Thus, using the electrochemical pulsed deposition

method, it is possible to obtain a structure including NiO nanoparticles with a narrow size

distribution arranged in the framework of CNTs. Furthermore, electrochemical pulsed deposition

prevents agglomeration of nanoparticles and makes it possible to control the film thickness by

varying the experimental parameters. Such an electrode exhibits an excellent stability, a high

surface coverage and an electrocatalytic activity toward the oxidation of insulin by various

electrochemical methods.

In order to further support the formation of NiONPs-MWCNTs composite, the X-ray diffraction

(XRD) profile of the prepared nanocomposite was also obtained and the result is shown in Fig. 1;

B (XRD data, supplementary material).

3.2. Study of electrochemical behavior of NiONPs/Nafion-MWCNTs

Electrochemical behavior of NiONPs/Nafion-MWCNTs was studied in 0.10 M NaOH solution.

In these voltammograms (supplementary material, S3), a pair of well-defined peaks is observed

for Ni oxide as was reported previously. These peaks are due to conversion of Ni(II)/Ni(III) to

each other in the alkaline solution via the following reaction (Asgari et al., 2011; Golikand et al.,

2006):

Ni(OH) 2 � NiOOH + e- + H+ (1)

The peak-to-peak potential separation (�Ep) in the voltammograms increased as the scan rate

was increased which shows charge-transfer kinetic limitations.

Sequential cyclic voltammograms for NiONPs/Nafion-MWCNTs/SPE modified electrode in

0.10 M NaOH solution is recorded (supplementary material, S4). In the early stages of potential

cycling, oxidation of Ni causes the appearance of a quasi-reversible redox peak. However, in the

Figure 1

later sweeps, the pair of peaks shift to more positive potentials due to a film growth and growth

of Ni(OH)2 on the electrode surface. Moreover, the charged nickel species, their corresponding

redox transition, and involvement of ionic species penetration into the film from the bulk of

solution, makes the film an ionic conductor (Majdi et al., 2007).

3.3. Electrocatalysis of insulin oxidation

The modifier layer of NiONPs/MWCNTs on the electrode surface acts as a catalyst for the

oxidation of insulin in alkaline solution. The inset of Fig. 2; A shows voltammograms recorded

for NiONPs/Nafion-MWCNTs/SPE in the absence (1) and in the presence of 0.5 mM insulin (2).

In the presence of insulin the NiONPs/Nafion-MWCNTs/SPE shows an increase in the anodic

peak current for peak (a) which followed by the appearance of a new peak (b) at more positive

potentials and a decrease of the cathodic peak current (c) during the reverse scan.

As shown in these voltammograms, the oxidation occurred in two regions of potential. In the

first region, Ni(�) species begin to produce (Ip,a). At upper potentials, insulin oxidation appears

as an increase in current (Ip,b) accompanied with a decrease in cathodic peak current in the

reverse scan (Ip,c). This shows clearly that the applied modifier in this process participate directly

in the electrocatalytic oxidation of insulin (Asgari et al., 2011; Golikand et al., 2006). In the

second region of potential, where Ni(�) species exist at the electrode surface as active catalysts,

the anodic peak (b) with a large peak current with respect to that of the former one (a) is

appeared. The appearance of this new anodic peak (b) leads to the conclusion that the insulin can

be adsorbed on Ni(III) when Ni(III) starts to form on the electrode surface at the potential of 700

mV. The adsorbed insulin oxidized under a chemical reaction by slow kinetics and produces

Ni(II) and other products.

This peak current (Ip,b) depends on the insulin concentration, addition of insulin in the electrolyte

results in conversion of Ni(III) to Ni(II) and then more amount of new produced Ni(II) oxidized

to Ni(III) again. The produced Ni(III) in these potentials oxidize the insulin and in the switching

potential, a great amount of Ni(III) are transformed to Ni(II), result in the decrease of the

cathodic peak (Asgari et al., 2011). Accordingly, the catalytic role of Ni(�) for insulin oxidation

can be shown as follows:

Peak (a) Ni(OH) 2 � NiOOH + e- + H+ (2)

NiOOH + Insulin � Ni(OH) 2 + Product (3)

Peak (b) Ni(OH) 2 � NiOOH + e- + H+ (4)

Peak (c) NiOOH + e- + H+ � Ni(OH) 2 (5)

3.4. Effect of scan rate

The effect of potential scan rate was investigated for different scan rates in the 0.10 M NaOH

solution in the presence of 0.50 mM insulin by cyclic voltammetry (supplementary material, S5).

The experiment result indicates that the insulin peak current increases linearly with the square

root of scan rates in the range of 10 � 400 mV s-1 (y = 6.7871x + 157.22) and with a correlation

coefficient of 0.998. This means that the electrocatalytic reaction of insulin at the modified

electrode is a diffusion controlled process. Moreover, the oxidation peak potential shifts

positively with increasing scan rate, suggesting a kinetic limitation in the reaction between the

NiO active sites and insulin (Salimi et al., 2003).

The relationship between the oxidation peak potential and scan rate is also investigated

(supplementary material, S5). Plotting the Epa vs. log��produces a straight line and the linear plot

can be expressed by the following equation:

E (V) = 0.11 log� (V s�1) + 1.16 (R2 = 0.993) (6)

According to Laviron’s equation (Laviron, 1979), the slop is equal to b/2 and b = 2.303RT/(1 �

�) n�F. Laviron derived general expressions for the linear potential sweep voltammetric response

of electroactive species at small concentrations (Laviron, 1979). The expressions for peak-to-

peak potential separations of �Ep > 200/n mV, where n is the number of exchanged electrons,

are as follows:

'0,

1 [ ]sp aE E X Ln

m��

� � (7)

� � � 1 1 1 /�s s s s s s s s p

RTLnk Ln Ln Ln Ln nF E RTnF

� � � � � � � �� � � � � � � �� � �

� (8)

where X = RT / (1-�s) nF, Y = RT / �nF, m = (RT/F) (ks/n�), Epa is the anodic peak potential and �s, ks, and � are the electron-transfer coefficient, apparent charge-transfer rate constant, and potential scan rate, respectively. From these expressions, as can be determined by measuring the variation of the peak potential with respect to the potential scan rate, s� and ks can be determined for electron transfer among the electrode and the surface-deposited layer by measuring the �Ep values. According to the results, it could be seen that for potential sweep rates of 200 to 500 mV s-1, the values of Ep are proportional to the logarithm of the potential sweep rate showed by Laviron. Use of the plot and Eqs. (7) and (8), the value of �s,a (anodic electron-transfer coefficient) was determined to be 0.74. Moreover, the mean value of ks was determined to be

2.57 s-1

.

3.5. Effect of varying concentration of insulin

Fig. 2; A shows the effect of the varying concentration of insulin (0.10 – 100 mM) on anodic

peak current at the modified SPE in 0.10 M NaOH solution. The anodic peak currents increased

linearly by increase the insulin concentration. The electrocatalytic currents level off at

concentration of ca. 50.0 mM, due probably to kinetic limitations (Salimi et al., 2003). The

Ni(III) species catalyzed the oxidation reaction of insulin so the reduction peak current decreased

with increasing insulin concentration. It can be assumed that the increase is due to the presence

of a diffusion-controlled process that appears to play an important role at low insulin

concentrations, while the insulin concentration increases, the whole oxidation process seems to

be catalytic in origin and its rate depends on the reaction between the insulin and Ni(III) species

present in the modifier film. By considering more sensitivity for hydrodynamic voltammetry,

calibration curve was obtained using amperometry in a stirring solution.

3.6. Chronoamperometry

Chronoamperometry was often employed to calculate some electrochemical parameters for the

diffusion-controlled electrode process (Majdi et al., 2007; Golabi et al., 2001; Asgari et al.,

2012; Sattarahmady et al., 2010). Fig. 2; B shows double-step chronoamperograms for the

NiONPs/Nafion-MWCNTs modified SPE in the absence and presence of different

concentrations of insulin. The applied potential steps were 700 and �200 mV. The current is

negligible when the potential is stepped down to �200 mV, indicating that the electrocatalytic

oxidation process is irreversible (Asgari et al., 2012). The current decayed as the reaction

proceeded to deplete the insulin in the diffusion layer of the electrode (Perenlei et al., 2011).

After the subtraction of the blank current (Ib) of the analyte current (Ia), catalytic current (Icat)

was obtained. The plot of Icat against t-1/2 along with the best fit for a 5.0 μM solution of insulin

was depicted in the inset (a) of Fig. 2; B. Under diffusion controlled process, a plot of Icat vs. t-1/2

will be linear.

In chronoamperometric studies, the current for the electrochemical reaction (at a mass transport-

limited rate) of an electroactive material (insulin in this case) that diffuses to an electrode with a

diffusion coefficient, D, is described by the Cottrell equation (Bard and Faulkner, 1980):

I = nFACb D1/2 /�� 1/2 t1/2 (9)

The currents decayed with time in a Cotrellian manner which was also confirmed by cyclic

voltammetry earlier. The diffusion coefficient of insulin can be estimated from the slope of the

plot of Icat vs. t-1/2, where A is the surface area of the working electrode, C is the bulk

concentration of insulin, D is the diffusion coefficient of insulin, other symbols have their usual

significance. In this work, A = 0.126 cm2, C = 5.0 × 10-6 M, n = 1 and the slope of the plot of Icat

versus t-1/2 is 6.887 �A s1/2. Thus, the value of D was calculated to be 4.20 × 10-5 cm2 s-1. The

initial current obeys Cottrellian behavior, but at longer times the current becomes steady state,

owing to edge effects influenced by the diffusion layers of neighboring electrodes (Widrig et al.,

1990).

Chronoamperometry can also be used to evaluate the catalytic rate constant according to (Bard

and Faulkner, 1980),

[Icat/Ib = �1/2[��/2 erf(�������exp(���������� (10)

Figure 2

where Icat and Ib are the currents in the presence and absence of the analyte, respectively, and � =

k�Ct is the argument of the error function. k� is the catalytic rate constant, and t is time. In cases

where � > 1.5, erf(���� is almost equal to unity, and Eq. (10) can be reduced to

Icat/Ib = �1/2 �1/2 = �1/2 (k�Ct ) 1/2 (11)

From the slope of the Icat/Ib versus t1/2 plot in Fig. 2; B, inset (b), the mean values of k� obtained

for insulin was 1.2 × 105 cm3 mol–1 s–1.

3.7. Amperometry

Fig. 3 shows the steady-state current response of insulin for the NiONPs/Nafion-MWCNTs

modified SPE with constant potential of 700 mV, in 0.10 M NaOH solution and rotation speed of

1000 rpm. As shown, during the successive addition of 1.0 μM of insulin solution (Fig. 4), a well

defined response is observed. The plot of response current versus insulin concentration is linear

over the wide concentration range of 20.0 – 260.0 nM(Fig. 4, inset). The calibration plot over the

concentration range of (13 points) has a slope of 1.83 �A/μM (sensitivity), correlation coefficient

of 0.997 and the detection limit of 6.1 nM at signal to noise ratio of 3. The detection limit, linear

calibration range and sensitivity for insulin determination at this modified electrode are

comparable with those obtained by using several modified electrodes (Table 1). The relative

standard deviation (RSD) for 4 measurements at 200 nM is 6.4%.

Figure 3

3.8. Stability and repeatability of the modified electrode

Stability tests were carried out for electrodeposited films under optimal conditions. Successive

potential scanning in 0.10 M NaOH solution in the presence of insulin showed that, after 20

cycles, there was a decrease of only <5% in the peak current and potential peak of insulin

electrooxidation remained almost unchanged (supplementary material, S6), indicating good

stability of the NiONPs/Nafion/MWCNTs modified electrode. This confirmed that the

intermediate and/or product of the electrooxidation reaction were not adsorbed at the

nanocomposite surface and it represented an anti-fouling behavior towards the insulin

electrooxidation (Sattarahmady et al., 2010).

Also, an insulin concentration of 0.50 mM in a 0.10 M NaOH solution was determined with 5

different electrodes. A reproducible current response with an excellent relative standard

deviation (RSD) of 5.7% was observed which demonstrated that the repeatability of the detection

of insulin concentration by this electrode was good.

The long-term stability of the modified electrode was also investigated by storing at room

temperature for 4 weeks. Only a small decrease of current (about 8.3%) for 0.5 mM insulin was

observed.

3.9. Electrochemical impedance spectroscopy

EIS is a powerful, nondestructive and informative technique that has played an essential role in

kinetic characterization and diagnosis of the events occurring at the different types of

electrode/solution interfaces in a wide range of applied DC potentials (Barsoukov and

Macdonald, 2005; Shervedani and Alinajafi-Najafabadi, 2011). Fig. 4 shows the Nyquist plots of

NiONPs/Nafion/MWCNTs/SPE recorded at 550 mV dc-offset, both in the absence (a) and

presence (b – j) of insulin in a 0.10 M NaOH solution. In this potential, system was controlled by

kinetics, and therefore, Rct was obtained from EIS experiments. In the absence of insulin, a

slightly depressed semi-circle was observed. Within the insulin concentrations ranging from 1.0

– 5.0 μM, a steady decrease in the diameter of the semi-circle was observed.

An equivalent circuit compatible with the results is designed (supplementary material, S7).. In

this electrical equivalent circuit, Rs, CPE and Rct are solution resistance, a constant phase element

corresponding to the double-layer capacitance and the charge-transfer resistance associated with

the oxidation of the insulin, respectively.

In order to obtain a satisfactory fitting of Nyquist plots, it was necessary to replace the double-

layer capacitance with a constant phase element in the equivalent circuit. The inset of Fig. 4,

shows insulin concentration dependency on Rct, where an initial sharp drop is terminated to a

very slow change as the concentration of insulin is increased above 2.0 μM.

4. Conclusion

A NiONPs/Nafion-MWCNTs modified SPE has been shown to be effective for insulin

determination, with a linear range from 20.0 until260.0 nM, and a detection limit of 6.1 nM.

Figure 4

Table 1

NiONPs were successfully electrodeposited onto the surface of Nafion-MWCNTs/SPE, and

characterized by using surface techniques and electrochemical methods such as SEM, EDX, EIS,

CV, CA and amperometry. The nano structure of NiO catalyst with consequent special properties

was confirmed and using pulse potential for electrodeposition of NiONPs was preferred to

conventional adsorption of NiO at the surface of electrode. The catalytic reaction of insulin at

NiONPs/Nafion-MWCNTs modified SPE was also studied by chronoamperometry and cyclic

voltammetry and the diffusion coefficient of insulin, D, and the electron-transfer coefficient, �,

were calculated as 4.2 × 10-5 cm2 s-1 and 0.74, respectively. The proposed modified electrode can

be prepared by a simple procedure with good reproducibility (RSD of 5.7%) and long-term

stability.

Acknowledgments

Financial support from the Research Affairs of Shahid Beheshti University is gratefully

appreciated.

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Figure captions

Fig. 1. TEM image of NiONPs/Nafion-MWCNTs (A); XRD spectrum of the NiONPs/Nafion-

MWCNTs (B).

0.100.10

Fig. 2. Cyclic voltammograms measured using a NiONPs/Nafion-MWCNTs/SPE in 0.10 M

NaOH solution containing: 0.1, 0.5, 1, 5, 8, 10, 50 and 100 mM insulin (A) ; Inset: Cyclic

voltammograms measured using a NiONPs/Nafion-MWCNTs/SPE in 0.10 M NaOH in the

absence (1) and presence (2) of 0.50 mM insulin. Chronoamperograms obtained for

NiONPs/Nafion/MWCNTs film modified SPE in the absence and in the presence of 5, 10, 15,

25, 50 μM insulin in 0.10 M NaOH using a potential step of 700 mV (B); Inset a: The linear plot

of Icat vs. t-1/2; Inset b: The linear plot of Icat/Ib versus t1/2.

Fig. 3. Amperometric response of rotating NiONPs/Nafion-MWCNTs modified SPE to insulin,

conditions 700 mV constant potential and stirring solution containing 0.10 M of NaOH,

successive addition of 1.0 μM. Inset: linear relationship between the peak current and

concentration of insulin.

Fig. 4. Nyquist diagrams of NiONPs/Nafion-MWCNTs/SPE in the absence (a) and in the

presence of 1.0 (b), 1,5 (c), 2.0 (d), 2.5 (e), 3.0 (f), 3.5 (g), 4.0 (h), 4.5 (i) and 5.0 (j) μM insulin

in 0.10 M NaOH solution . DC potential was 550 mV. Frequency ranges of 10 kHz to 100 MHz.

Symbols are the experimental data and lines show the approximated results; inset: Dependency

of Rct values on insulin concentration derived from the data of Nyquist diagrams.

Table 1 Analytical parameters for detection Insulin at several modified electrodes

Electrode Method Dynamic range Sensitivity LOD Reference

SPEa/MWCNTb/NiONPsc Amperometry 20 nM � 0.26 μM 1.83 �A/μM 6.1 nM This work

SPE/CNT/NiCoO2 Amperometry 0.17 � 75 μg/mL 22.6 μA/mg mL 1.06 μg/mL (Arvinte et al., 2010)

GCEd/CNT Amperometry FIAe 100 � 1000 nM 48 nA/μM 14 nM (Wang and Musameh, 2004)

GCE/MWCNT/DHP Amperometry FIA 0.8 � 2.5μM 1.33 nA/μM 1μM (Snider et al., 2008)

GCE/CNT CV 20 � 400 ng/mL 1.85 μA/μg mL 7.75 ng/mL (Wang and Li, 2009)

CCEf/Nickel powder-K4[Mo(CN)8] Amperometry 0.5 � 500 nM 6.14 μA/μM 0.45 nM (Salimi et al., 2006)

GCE/Guanine/NiONPs Amperometry 0.1 � 4000 nM 101 μA/μM 22 pM (Salimi et al., 2008)

CCE/[Ru(bpy)(tpy)Cl]PF6 Amperometry 0.5 � 850 nM 7.60 μA/μM 0.4 nM (Salimi et al., 2003)

GCE/IrOx Amperometry 50 � 500 nM 35.2 nA μM 20 nM (Pikulski and Gorski, 2000)

Carbon microelectrode/RuO/RuCN Amperometry � 441 μA /μM 500 nM (Kennedy et al., 1993)

GCE/Chitosan/MWCNT Amperometry 100 � 3000 nM 135 nA/μM 30 nM (Zhang et al., 2005) a Screen printed electrode. b Multi-walled carbon nanotubes. c Nickel oxide nanoparticles. d Glassy carbon electrode. e Flow injection analysis. f Carbon ceramic electrode.

Highlights

� An amperometric sensor for insulin, using screen printed electrode is designed. � NiO nanoparticles are generated on the MWCNTs/SPE using pulsed electrodeposition. � At low concentrations, the mechanism relies on diffusion through the bulk. � The behavior of the proposed electrode is studied by amperometry, CA, CV and EIS.

Fig. 1

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Figure(s)

Fig. 2

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Fig. 32

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Fig. 43

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Fig. 54

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