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Sensors and Actuators B 150 (2010) 756–763

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journa l homepage: www.e lsev ier .com/ locate /snb

lectrocatalytic oxidation and determination of insulin at CNT-nickel–cobaltxide modified electrode

dina Arvinte, A. Caroline Westermann, Adama Marie Sesay ∗, Vesa Virtanenaboratory of Biotechnology, Kajaani University Consortium, University of Oulu, Salmelantie 43, 88600 Sotkamo, Kainuu, Finland

r t i c l e i n f o

rticle history:eceived 10 May 2010eceived in revised form 2 August 2010

a b s t r a c t

CNT-nickel–cobalt oxide/Nafion composites were prepared by combining the nickel–cobalt oxide powderwith CNT directly into the Nafion solution used as a binder and deposited on screen-printed elec-

ccepted 3 August 2010vailable online 11 August 2010

eywords:ickel–cobalt oxideNT

trodes substrates. Their electrochemical behaviour was investigated by means of cyclic voltammetryand amperometry techniques in NaOH and phosphate buffer solutions. The electrocatalytic activity ofthese sensors was evaluated in terms of sensitivity, detection limit and stability towards insulin detection.CNT-nickel–cobalt oxide/Nafion electrodes enable the oxidative detection of insulin in aqueous solutionat physiological pH, with a good sensitivity (22.57 �A × mg−1 × mL−1). An electrochemical activation stepof the sensor in alkaline solution has been shown to be necessary for a three times more sensitive response

e-co

nsulin oxidation of CNT-nickel–cobalt oxid

. Introduction

Insulin is one of the most intensely studied and well-haracterized protein molecules to date. The insulin moleculeonsists of two polypeptide chains linked by two disulphideridges, an A chain of 21 amino acid residues and a B chain of0 amino acid residues. From the determination of the termi-al residues of insulin it was suggested that the submolecule ofolecular weight 12,000 is made up of four open peptide chains

ound together by disulphide linkages [1]. Many methods haveeen proposed and investigated for determination of insulin hor-one in biological fluids. The immunoassay of insulin has beenidely used for determination of insulin, but cross reaction andon-specific binding with the coexistent biomolecules againstnti-insulin antibody are the major interferences, decreasing theccuracy of determination of insulin in biological samples [2].

Electrochemical detection of insulin is of great interest as it canrovide good sensitivity and also reduced analysis times, whichnable the continuous real-time measurements. A large variety oflectrodes modified by redox mediators and electrocatalysts haveeen proposed for promoting the oxidation and detection of insulin.n insulin-sensitive electrode modified with a ruthenium diox-

de/cyanoruthenate (RuO/CN-Ru) has been developed by Cox andray [3]. This catalyst film accelerates the oxidation of insulin incidic media. More convenient for the monitoring of insulin athysiological pH are the ruthenium oxide (RuOx) [4], iridium oxide

∗ Corresponding author. Tel.: +358 86332 1318;fax: +358 86332 1313.E-mail address: adama.sesay@oulu.fi (A.M. Sesay).

925-4005/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.snb.2010.08.004

mposite electrode toward insulin detection.© 2010 Elsevier B.V. All rights reserved.

(IrOx) [5], and ruthenium metallodendrimer [6] catalytic layers.However, high cost and scarce source of noble metal oxides lim-its their application. Recently more and more interest has beenfocused on cheap transition metal oxides, such as nickel oxide (NiO)and cobalt oxide (CoO). Regarding the oxidation of insulin, an elec-trode based on nickel oxide–carbon composite has been developedas an amperometric detector for hydrodynamic amperometry andflow injection analysis of insulin [7].

Cobalt oxide has been studied as a catalyst several times over theyears. Cobalt oxide has been used and investigated for its activity forCO and hydrocarbon oxidation [8–11] and also for electrocatalyticoxidation of carbohydrates [12].

The alternatives to improve the activity of a mono-oxide arerather limited. So mixed oxides are often used to modulate theelectrode properties through synergistic effects arising from theintimate electronic interaction of the components [13–15]. Mixedoxide electrode materials are involved in analytical applicationsdue to the appropriate compromise between electrocatalytic activ-ity, long-term stability and cost.

One of the most promising materials based on transition metaloxides, frequently used in an alkaline electrolyte, is the mixedNi and Co oxide which is, moreover, a stable and cheap elec-trode material. It was proved that the addition of cobalt to nickeloxide/hydroxide enhances the electrochemical performance of theelectrodes [16,17] and the major beneficial effect of adding cobalt

is the increase in electrode conductivity.

Mixed Ni and Co oxide films have been extensively studiedin the past, primarily due to their numerous promising applica-tions. These include their use as alternative electrode materialsfor supercapacitors [18–22]. Ni and Co oxides exhibit interesting

Actuators B 150 (2010) 756–763 757

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lectrocatalytic activities, which make them attractive for O2 evo-ution [23,24]. A different combination of the two oxides, NiCo2O4pinel, is also known to be an active and stable bifunctional catalystor oxygen evolution and reduction in alkaline media, as well as aatalyst in alcohol and amine electro-oxidation [25–27]. There areonfirmations in the literature that mixing Ni and Co oxides bringseneficial effects on their electrocatalytic activity, but most of thetudies were directed toward the electrocatalytic activity investiga-ions of this catalyst of mixed oxides for both oxygen reduction andvolution reactions [24,26,28–30]. There is a challenge for usingixed metal oxide (NiO and CoO) for oxidation of biologic com-

ounds, as insulin.The electrocatalytic behaviour of metal oxides materials can be

urther improved by the presence of carbon materials, which con-ribute to the total specific surface area and porosity of the material.ecently, special attention has been given to chemically modifiedarbon electrodes and carbon composite electrodes to improve thelectrocatalytic activity for the determination of different analytes.hemically modified electrodes are mainly prepared by electro-hemically depositing a catalyst onto carbon electrode surfaces,hile carbon composite electrodes are usually formed by mixing

atalysts with carbon powder [31,32].In the present work, we report the preparation of the CNT-

ickel–cobalt oxide-composite electrodes by simply composingi–Co oxide powder together with CNT into the pore of Nafionembrane and the investigation of their electrochemical prop-

rties. In addition, the influence of loading mass of Ni–Co oxiden electrochemical properties for the composites was reported.o evaluate the utility of composite based electrode for analyti-al applications they have been used for amperometric detectionf insulin, an important polypeptide hormone which regulates theetabolism of glucose. The electrochemical detection of insulin at

lassy carbon electrodes modified by carbon nanotubes [33] or bylms of chitosan and multiwall carbon nanotubes [34] was investi-ated and reported before in the literature. However, the potentialpplication of Ni–Co mixed oxide combined with CNT as the elec-rode materials for insulin detection has never been discussed.

. Experimental part

.1. Reagents

Sodium phosphate (Na2HPO4 and NaH2PO4) and KClsed for buffer solution preparation were supplied by Merckwww.merck.com). Multiwall carbon nanotubes (MWNT, 6–13 nmiameters and 2.5–20 �m length, 99.8% purity) were purchasedrom Sigma–Aldrich (www.sigmaaldrich.com) and used aseceived, without any further purification. The Nafion 5% solutionas provided by Fluka. Nickel and cobalt mixed oxide nanopow-er (NiCoO2, <150 nm, 99%), insulin 10 mg/mL stock solution andodium hydroxide pellets (NaOH) were supplied by Sigma–Aldrich.

.2. Apparatus and methods

Cyclic voltammetry and amperometry methods were used tonvestigate the electrocatalytic properties of the nickel–cobaltxides composite. All experiments were carried out using aotentiostat PalmSens Instrumentation (Palm Instrument BV, theetherlands, www.palmsens.com) connected to a PC. The electro-hemical measurements were carried out in phosphate buffer (PBS).1 M with KCl 0.1 M, pH 7.5 (unless otherwise stated) in a conven-

ional electrochemical cell of 3 mL volume, using a three-electrodeystem with a planar configuration of a screen-printed electrodeSPE), fabricated and purchased from Biosensor laboratory, Uni-ersity of Florence, Italy (www.ebsr.it). The working carbon basedlectrode is of 3 mm diameter.

Fig. 1. Cyclic voltammograms in 1 M NaOH for (a) CNT/Nafion, (b) NiCoO2/Nafionand (c) CNT-NiCoO2/Nafion electrodes (Nafion 1.7%; 3 �g NiCoO2/electrode). Scanrate: 0.05 V/s.

For the amperometric measurements, the sensors wereimmersed in the stirred buffer solution, applying the proper poten-tial. When a stable baseline was reached, the proper volume ofinsulin (to give a certain final concentration) was added, followedby recording the steady-state current.

All solutions used in this work were prepared with double dis-tilled water. All electrochemical experiments were carried out atroom temperature and the potentials were referred to Ag/AgCl.

2.3. Preparation of mixed oxide composite material

The Nafion-bonded electrodes were prepared by mixing theappropriate volume of Nafion of an optimum concentration andNiCoO2 catalyst in a small bottle, stirred and dispersed withthe aid of an ultrasonic bath. 2 �L from resulting mixture weredropped onto the SPE working surface. For the preparation ofCNT-NiCoO2 composites electrodes, the CNT powder was addedto NiCoO2/Nafion solution to obtain a concentration of 8 mg/mL forCNT. The electrodes were allowed to dry for 1 hour at room tem-perature then kept at 4 ◦C until used. All measurements were madein triplicate unless otherwise stated.

3. Results and discussions

3.1. Cyclic voltammetry studies of CNT-nickel–cobalt oxidescomposites in alkaline medium

The electrochemical behaviour of the CNT-nickel–cobaltoxide/Nafion composite electrodes was characterized by differentelectrochemical measurements in 1 M NaOH aqueous solutions.The potential was swept in a large window potential with a scanrate of 0.05 V/s. To further improve the electrochemical behaviourof the sensor, the mixed metal oxide have been combined with CNT,in a ratio of 16:3 CNT:NiCoO2 (w/w). The typical cyclic voltam-mograms of CNT/Nafion, NiCoO2/Nafion and CNT-NiCoO2/Nafionelectrodes are shown in Fig. 1.

In Fig. 1 curve (a), it can be seen that the CNT electrode showsa featureless cyclic voltammogram, characteristic for a typicaldouble-layer capacitive behaviour. Curve (b) is the cyclic voltam-mogram of NiCoO2/Nafion electrode, which shows very weakoxidation and reduction peaks at 0.5 and 0.45 V, at a scan rate of

50 mV/s.

The absence of two separate oxidation peaks for cobalt oxide andnickel oxide has been previously reported [35,16] and explainedby the fact that the Co oxide redox chemistry occurs in the samepotential range as the Ni(II)/Ni(III) transition and therefore cannot

758 A. Arvinte et al. / Sensors and Actuators B 150 (2010) 756–763

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ig. 2. Cyclic voltammograms for CNT-NiCoO2/Nafion electrodes (Nafion 1.7%; 3 �giCoO2/electrode) in 1 M NaOH at different scan rates: 10, 20, 30, 40, 50, 100, 150nd 200 mV/s.

e resolved separately. According to literature, for Ni:Co molar ratiof 1:1, the two pairs of peaks corresponding to Ni and Co respec-ively overlap and become one pair of sharp peaks [36]. Our results in agreement with that reported in literature.

The obtained pair of current peaks corresponds to the surfacearadaic reactions according to concomitent reactions (1) and (2):

iO + OH− → NiOOH + e− (1)

oO + OH− → CoOOH + e− (2)

here the proton diffusion and charge transfer are both con-ributing to the total electrode reaction [37]. Eq. (1) describes thexidation reaction of Ni2+ (NiO or/and Ni(OH)2) to Ni3+ (NiOOH)n which nickel hydroxide can form at the surface of nickel oxideuring cycling in NaOH solution [37,38]. Eq. (2) corresponds forhe analogous transformation of Co2+ (oxide or hydroxide) to Co3+

CoOOH) [12,36,39].However, for CNT-NiCoO2/Nafion electrode (16:3 CNT:NiCoO2

atio), large oxidation and reduction peaks are clearly observedt 0.25 and 0.14 V, respectively (curve c). Compared with theiCoO2/Nafion electrodes, the anodic and cathodic peak poten-

ials of the CNT-NiCoO2/Nafion electrode are shifted in the negativeirection. This is in agreement with that reported by Vitanov andoworkers [26]. It is well emphasized that the current values andoltammetric profile of the CNT-NiCoO2 composite electrode areuch higher and better than those of NiCoO2 electrodes without

NT. This is probably due to that CNT entangle with NiCoO2 and pro-ide unobstructed pathways for OH− transport during the redoxrocess. In addition, high conductivity of the CNT also facilitatesransportation of electrons during rapid oxidation/reduction pro-ess. The benefits of using CNT are based on the special structuref CNT, which offer good electronically conducting pathways to theulk and surface of NiCoO2.

Typical cyclic voltammograms for CNT-NiCoO2 composite elec-rode at various scan rates are shown in Fig. 2. As scan rate increases,nodic peak potential and cathodic peak potential shift in the morenodic and more cathodic direction, respectively. The potential dif-erence between Epa and Epc is relatively constant at low scan rates,nd then increases at high scan rate. Consequently, on the basisf liquid electrolyte reversibility criteria, we can assume that theeaction approaches reversibility only at low scan rates [40].

Characteristic parameters of cyclic voltammetry obtained fromig. 2 are shown in Fig. 3 as a function of scan rate, v. The voltammet-ic peak currents ipa and ipc are plotted against square root of scanate, v1/2 (Fig. 3A) and the peak potentials Epa and Epc are plottedgainst v (Fig. 3B). In our study, the linear relationship between ipa,

Fig. 3. Variation of anodic and cathodic peak currents vs. square root of scan rates(A) and variation of peak potentials vs. scan rates (B).

respectively ipc and v1/2 suggests that the oxidation of NiCoO2 is dif-fusion limited, as stated also by other authors [41]. In addition, withincreasing the scan rates, the peak separations begin to increase,indicating the limitation arising from charge transfer kinetics.

3.2. Cyclic voltammetry studies of CNT-nickel–cobalt oxidescomposites in neutral medium

According to the literature [42], only at very high potential(>1.5 V), Co2+ and Ni2+ can be oxidized to high valent oxides. More-over, Ni and Co oxide-based materials, when used as heterogeneouscatalysts, show good chemical stability and electroactivity only inalkaline conditions [13]. In Fig. 4A, the cyclic voltammetry responseof CNT-NiCoO2 composite electrode in NaOH has been comparedto that obtained in phosphate buffer solution (PBS) pH 7.5 at a lowscan rate.

The profile of cyclic voltammogram is completely shifted in ahigher range of potential when the experiment is performed inphysiological pH. The oxidation peak is well defined with a peakpotential of 0.8 V, but the reduction peak is diminished in current,appearing at 0.65 V. As presented in Eqs. (1) and (2), the Faradaicreactions taking place at electrode surface involves the OH− groups,consequently, they are pH dependent. The potential for NiOOHand CoOOH formation increases by decreasing the hydroxide con-

centration, which is in agreement with reported pH dependentbehaviour for nickel oxide [43] and cobalt oxide [44].

For comparison, the electrochemical behaviour ofNiCoO2/Nafion and CNT/Nafion electrodes were also investi-gated in PBS pH 7.5 and presented in Fig. 4A as cycles c and d,

A. Arvinte et al. / Sensors and Actuators B 150 (2010) 756–763 759

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ig. 4. (A) Cyclic voltammograms for CNT-NiCoO2 composite electrode (Nafion 1.7%nd (d) are cyclic voltammograms in PBS pH 7.5 for NiCoO2/Nafion and CNT/Nafionnd (C) in PBS pH 7.5. Scan rate: 20 mV/s.

roving again the synergistic effect between the two components,ixed metal oxide and CNT.Repetitive cyclic voltammetry measurements were performed

o investigate the stability of CNT-NiCoO2 composite in bothaOH and PBS pH 7.5 media. Fig. 4B shows 10 consecutive cyclicoltammograms of CNT-NiCoO2 composite electrode in NaOH 1 Metween the potential of -0.2–0.6 V at a scan rate of 20 mV/s. Theery slow shift of oxidation peak potential observed during electro-hemical cycling suggests a gradual conversion of Ni2+ to Ni3+ ando2+ to Co3+, forming the oxide hydroxide layer. Fig. 4C presents 10onsecutive cyclic voltammograms of CNT-NiCoO2 electrode in PBSH 7.5, demonstrating the very stable behaviour of the composite

n physiological pH media.

.2.1. Optimization of nickel–cobalt oxides loading in theomposite

The effect of loading mass of the nickel–cobalt oxide materialnside the CNT-NiCoO2/Nafion composite on the electrochemical

ig. 5. The effect of NiCoO2 loading in the composite material on electrochemicalehaviour of the CNT-NiCoO2-based electrode: (a) 0 �g; (b) 3 �g; (c) 6 �g; (d) 9 �gnd (e) 16 �g NiCoO2 on the working electrode surface. Scan rate = 10 mV/s. PBS pH.5.

NiCoO2/electrode) performed in (a) 1 M NaOH and (b) 0.1 M PBS pH 7.5. Scans (c)rodes. (B) 10 consecutive scans for CNT-NiCoO2 electrode performed in 1 M NaOH

behaviour of electrodes was investigated by means of cyclic voltam-metry in PBS pH 7.5 and the corresponding results are shown inFig. 5. Five different electrode configurations have been preparedby varying the ratio of CNT:NiCoO2 from 8:0 to 8:1.5, 8:3, 8:4.5 and8:8. The final amount of nickel–cobalt oxide material existing ontothe electrode varies from 0 to 16 �g.

With the increase of the content of NiCoO2 in CNT-metaloxides/Nafion composites, the peak currents increase and thevoltammetric behaviour is improved. This can be explained by thefact that carbon nanotubes provide a large surface for metal oxidedeposition on electrodes and thus providing a synergistic relation-ship for an improved electron transfer between the base electrodeand the CNT-NiCoO2 modifying material. Second, uniform disper-sion of the cross-linked CNT among the NiCoO2 resulted in goodinterconnection of metal oxide at the surface and interior of theelectrodes, forming a homogenous network which facilitates thetransport of hydroxyl during the redox process.

It is observed that the oxidation peak is proportional to the load-ing mass of NiCoO2 in the range from 3 to 9 �g/electrode. Then,increasing the NiCoO2 mass to 16 �g/electrode, the electrochemicalresponse is rather low. This result is sustained by the amperomet-ric response obtained for insulin with these five different electrodeconfigurations and presented in Table 1. For further work, compos-

ite containing 8:4.5 ratio between CNT:NiCoO2 has been chosen formodification of the electrodes.

Table 1Analytical parameters of CNT-NiCoO2 composite electrode with different loading ofmetal oxide material.

CNT:NiCoO2 Sensitivity(�A mg−1 mL−1)

LOD (�g/mL) Linear range(�g/mL)

8:0 10.76 ± 1.34 2.78 1.2–758:1.5 14.48 ± 1.41 2.07 1.2–75

8:3 17.68 ± 2.10 1.35 0.33–758:4.5 22.57 ± 2.35 1.06 0.17–75

8:8 16.52 ± 2.81 1.45 0.33–540:3 0.76 ± 0.07 31.5 2.6–54

760 A. Arvinte et al. / Sensors and Actuators B 150 (2010) 756–763

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ig. 6. Voltammetric response of (A) CNT/Nafion electrode , (B) NiCoO2/Nafion elect.5 in the absence (a) and presence (b) of 45 �g/mL insulin. Scan rate: 10 mV/s.

.3. Insulin detection on CNT-nickel–cobalt oxides composites ineutral medium

The objective of this work was to fabricate a modified elec-rode capable of the electrocatalytic oxidation of insulin. Thebility of CNT combined with mixed Ni and Co oxides catalysts tolectro-oxidize insulin was investigated by different electrochem-cal measurements in 0.1 M PBS pH 7.5 solution in the absencend presence of insulin at 10 mV/s vs. Ag/AgCl. For comparison,lectrodes based on only CNT or NiCoO2 respectively, have beenvaluated for insulin oxidation in the same conditions.

As seen in Fig. 6A, at the surface of CNT/Nafion electrode, oxi-ation of insulin starts above +0.5 V and the current rises up to0.9 V. A small response was observed for insulin oxidation atiCoO2/Nafion electrodes (Fig. 6B), where a slight increase of theeak current (shifted to +0.9 V) carried out in the presence of

nsulin. In contrast, as seen in Fig. 6C, for electrodes modifiedith CNT-NiCoO2/Nafion, upon the addition of 45 �g/mL insulin,

here is a significant enhancement of anodic peak current, indicat-ng a strong catalytic effect of modified electrodes. Also the peakotential appeared for oxidation of insulin is lowered from 0.9 V to.8 V, emphasizing the benefit of synergy existent between the twoomponents. Subsequent amperometric detection work employeddetection potential of +0.8 V that offered the most favourable

ignal-to-noise characteristics.

We can state that the enhanced analytical signals of CNT-Ni–Co

xide-based electrode with respect to CNT and Ni–Co oxide elec-rodes can be attributed to a larger amount of mixed metal oxideeposited on the CNT surface due to their larger surface area andherefore, a larger amount of metal oxide exposed to the solution.

nd (C) CNT-NiCoO2/Nafion composite electrode (9 �g NiCoO2/electrode) in PBS pH

This hypothesis is in agreement with other works reported in liter-ature concerning the composite of metal oxide and CNT [45].

The stability and antifouling properties of the modified elec-trode toward insulin oxidation were tested by evaluating thedecrease of the electrocatalytic oxidation current of insulin dur-ing successive potential cycling of the modified electrode. From thefirst cycle to the second one, the modified electrode showed a 10%decrease of the oxidation current, and then for next 9 cycles thepeak currents were stable (data not shown). This behaviour indi-cates that the CNT-NiCoO2/Nafion modified electrode surface is notblocked by insulin or its oxidation products.

3.4. Amperometric detection of insulin on CNT-NiCoO2 compositeelectrodes in physiological pH

Similar to voltammetric experiments, the effect of loading massof mixed oxide material inside the CNT-NiCoO2/Nafion compositeelectrodes was investigated by means of amperometric detectionof insulin in PBS, pH 7.5. Electrodes have been tested for successiveadditions of insulin in stirred solution, applying a +0.8 V potentialvs. Ag/AgCl until the saturation plateau was reached. The measuredcurrents increase, as the insulin concentration in cell solution isincreased and then the amperometric current have been plottedvs. insulin concentrations (data not shown). The equations obtainedfor each electrode configuration are extracted and summarized in

the Table 1.

As reported in Table 1, the sensitivity of electrodes increaseswith increasing the content of NiCoO2 in the resulting compositematerials. However, further increase of the NiCoO2 content over9 �g/electrode did not affect in a better way the sensitivity of the

A. Arvinte et al. / Sensors and Actuators B 150 (2010) 756–763 761

Fig. 7. Voltammetric response to 33 �g/mL insulin obtained for (a) treated CNT-NiCoO2 electrode and (a′) non-treated CNT-NiCoO2 electrode. (b) and (b′) arevTuC

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strong electrocatalytic activity to 0.1 mM ascorbic acid (additionb) and quite significant activity toward uric acid and paracetamol.This result indicates that the Nafion present in the composite filmcannot completely block the diffusion of ascorbate and urate andparacetamol into the composite films.

oltammograms recorded in PBS pH 7.5 for both electrode types; scan rate: 10 mV/she inset shows the amperometric responses to successive additions of insulinntil 80 �g/mL final concentration at (a) treated CNT-NiCoO2 and (a′) untreatedNT-NiCoO2 electrode; E = 0.8 V.

omposite electrode for insulin detection. The NiCoO2-based elec-rodes, in contrast, exhibit a very low sensitivity for detection ofnsulin at physiological pH.

.5. Improvement of the CNT-NiCoO2 composite electrodesensitivity for insulin detection

Substantial improvement in the performance of amperomet-ic sensors for insulin is achieved by subjecting the CNT-NiCoO2omposite electrodes to an electrochemical pretreatment in alka-ine media prior to the detection of insulin in physiological pHuffer. For this purpose, electrodes modified with CNT-NiCoO2ere exposed to potential cycling in 1 M NaOH solution, for a num-

er of 5 scans. The potential was swept between -0.2 V and 1.1 V,ith a scan rate of 50 mV/s. The electrodes were then rinsed thor-

ughly with distilled water and tested for voltammetric or/andmperometric response to insulin in PBS solution at physiologic pH.hese electrodes were compared to similar electrodes that were notretreated in alkaline media, as previously reported.

Fig. 7 emphasizes the differences between voltammetric andmperometric responses to insulin oxidation obtained at pre-reated and non-treated electrodes. It has been found that thelectrochemical alkaline pretreatment of electrodes results in aignificantly improvement of the response to insulin.

The sensitivity of electrodes for insulin detection is increasedrom 22.57 ± 2.35 to 66.48 ± 6.52 �A mg−1 mL−1 through the elec-rochemical pretreatment performed in alkaline media. Themperometric response of these electrodes is linear in a lower rangef insulin concentration from 0.1 until 31.5 �g/mL. The favourableignal-to-noise characteristics of pretreated electrodes contain-ng 8:4.5 ratio of CNT and NiCoO2, indicate a detection limit of.22 �g/mL, which correspond to a 38 nM concentration (for sig-al/noise = 3). Other metal-oxide-based insulin sensors reported

n literature offered detection limits of 95 [3], 23 [4], 20 [5] andnM [6]. The stabilization of the baseline in amperometric exper-

ments is faster for treated electrodes, and also, the response times improved.

It is believed that NiO in contact with an alkaline solutionends to change to nickel hydroxide at the surface [38,41,44] form-

ng a thin surface layer onto NiO. The same process happens tooO, which forms a cobalt hydroxide layer [39]. Consequently, theoltammetric response will be more significant as such in the reac-ion (1) will be involved NiO and Ni(OH)2 and in reaction (2) wille CoO and Co(OH)2.

Fig. 8. Stability of the CNT-NiCoO2 electrode response to 33 �g/mL insulin in PBS pH7.5 for (a) non-treated electrode (b) pretreated electrode in alkaline solution beforethis experiment. Operating potential: +0.8V; stirring rate: 400 rpm.

Operational stability of the CNT-NiCoO2 composite electrodeshave been evaluated by measuring the repetitive response to33 �g/mL insulin in a batch amperometric experiment, applyinga potential of +0.8 V. Between measurements the electrodes wererinsed with distilled water. As expected, the response of treatedelectrodes is higher than that of non-treated electrode, but theoperational stability is satisfactory for both cases, as shown in Fig. 8.The average response for non-treated electrodes is 397 nA with astandard deviation of 45, while for pretreated electrode the meanvalue of the response is 1165 nA with a standard deviation of 103.

3.6. The selectivity of CNT-NiCoO2 composite electrodes forinsulin detection

One of the most important aspects to take into account forany analytical application of sensors is the study of the effect ofinterfering compounds potentially present in the real sample. Inorder to evaluate the possible interfering effect of few commonsubstances found in biologic fluids, the amperometric response ofthe CNT-NiCoO2 electrode was measured for successive additionsof 16.6 �g/mL insulin (corresponding to 2.7 �M) and 0.1 mM ofascorbic acid, uric acid and paracetamol (acetaminophen) under theoptimized experimental conditions (Fig. 9). It can be observed thatthe CNT-NiCoO2/Nafion composite electrode (dotted line) has very

Fig. 9. Dotted line: Influence of electroactive interferents on CNT-NiCoO2 electrodein insulin solution 16 �g/mL (a). 0.1 mM of (b) ascorbic acid, (c) uric acid and (d)paracetamol in PBS pH 7.5, stirred solution, applied potential 0.8 V. Solid line is forCNT-NiCoO2 electrode covered with a second layer of Nafion.

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A very common method used for diminution or eliminationf interferences is to cover the electrode surface with an addi-ional membrane for blocking the permeation of interferents intohe biosensor catalytic layer. Among other films preventing thenterferents based on electrostatic repulsion, Nafion is a negatively-harged perfluorinated ionomer that can effectively restrict thenionic interferents from adhering on its surface. It was confirmedhat the Nafion coating prevents interference of anionic substancesuch as ascorbic acid and uric acid, and decreases acetaminophennterference [46,47]. A favorable effect was achieved when elec-rodes are coated with a combined film of cellulose acetate andafion, which eliminates ascorbate completely, and three layerslso eliminate acetaminophen response [48].

A first attempt in this work was to cover the surface of CNT-iCoO2/Nafion electrode with a second layer of 2 �L Nafion 2% and

hen the electrode was treated in alkaline solution as explainedefore and tested for insulin and interferents in similar condi-ions as in previous experiments. In Fig. 9 (solid line) is shownhat the response to 16.6 �g/mL insulin is reduced with only 15 nAhich corresponds to 2% diminution of the sensitivity for insulin. In

ontrast, the response to ascorbic acid at the Nafion-covered elec-rode is significantly diminished (∼50%). A similar situation can alsoe observed for paracetamol (∼60%), whereas the interference ofric acid is very much reduced (∼80%). These results indicate thathe Nafion membrane-covered electrodes can efficiently reducehe interference of uric acid and paracetamol. Further study is inrogress for a better diminution of ascorbic acid interference.

Level of ascorbic acid and paracetamol in biological fluids aretrongly dependent of the intake. Reported studies have confirmedhat oral or intravenous administration of acetaminophen in nor-

al human subjects produces maximum plasma concentration in0–50 min, and the peak plasma concentration rests in the range of.14 mM when the maximum therapeutic dose (1000 mg) is intro-uced [49,50]. Serum levels of ascorbic acid increased significantlyfter vitamin C ingestion. Therefore, the interference of ascorbiccid and paracetamol can be avoided when tests for insulin detec-ion in physiological fluids are intended.

. Conclusions

Electrochemical techniques were applied in connection withcreen-printed electrodes to study new composite materialsbtained by combining the high surface area carbon nanotubesMWNT) with conductive mixed metal oxides (NiCoO2) and theirlectrocatalytic activity toward detection of insulin. It has beenhown that the electrocatalytic activity toward insulin detections improved when metal oxides are composited with carbon nan-tubes material compared to only metal oxide modified electrodesr only CNT based electrodes. These results indicate the muchigher electrochemical reversibility of the oxide-nanocompositeaterial than simply metal oxide material due to the entangled

etwork and porous structure of CNT, which form a well accessiblelectrode/electrolyte interface and accelerates the charge trans-er process. The oxide-nanocomposite based electrodes showedxcellent electrocatalytic activity toward insulin electro-oxidationn physiological pH solution. The loading mass of Ni–Co oxiden the composite material has been optimized and electrodes

ith 9 �g NiCoO2/electrode (composite containing 8:4.5 ratio ofNT:NiCoO2) show the best electroanalytical properties for insulinxidation. Amperometric detection of insulin has been performed

t pH 7.5, applying 0.8 V potential for CNT-NiCoO2-modified elec-rode. An electrochemical pretreatment in alkaline media prioro the detection of insulin in physiological pH buffer proved toncrease 3 times the sensitivity of electrodes for insulin. Under theptimized conditions, the calibration curve is linear in the concen-

[

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ators B 150 (2010) 756–763

tration range 0.1–31.5 �g/mL, with a good sensitivity allowing adetection limit (signal/noise = 3) of 0.22 �g/mL. The very sensitiveand stable response for insulin at a CNT-NiCoO2-modified electrodeindicated that it can be used to detect insulin at concentrations aslow as 0.22 �g/mL, in physiological pH, showing great promisesfor applications of these electrodes in biological samples of clin-ical interest. An additional coating of Nafion has effectively beenused to reduce the interference of uric acid and paracetamol in thedetermination of insulin.

Acknowledgements

The work has been partially funded by the MeasurepolisResearch Consortium (MeRC) program, Kainuu Finland.

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Biographies

Adina Arvinte completed her PhD degree in analytical chemistry at University ofBucharest, Romania in 2009. She is currently a post-doctorate researcher at the Lab-oratory of Biotechnology, Sotkamo, a unit of University of Oulu, Finland. Her researchinterests include functional nanocomposite materials, the design and application ofnew electrochemical sensors and biosensors.

Caroline Westermann is a MSc student at the University of Hohenheim, Germanyand a visiting student at the Laboratory of Biotechnology in Sotkamo, University ofOulu, Finland in 2009.

Adama Marie Sesay completed her PhD degree in Biosciences from Cranfield Uni-versity in 2003 and has carried out her post-doctoral fellowship at the DanishTechnological Institute, Denmark. She is currently a project manager and seniorscientist at The Laboratory of Biotechnology, Sotkamo, University of Oulu, Fin-land. Her main research focuses are in biosensing applications, micro-fluidics andmicro/nanotechnology.

Vesa Virtanen obtained his PhD degree from University of Oulu in 1993. He carriedout his post-doctoral fellowship at European Union Joint Research Centre, Insti-tute of Reference Materials and Measurements (IRMM) Belgium during 1994–1996.He has operated as scientific officer for European Commission during the years1996–1999 at IRMM. He has been a visiting scientist at University of Tübingen,

development. Currently he is the head of the Laboratory of Biotechnology, Sotkamo,a unit of University of Oulu and he is an adjunct professor within the ChemistryDepartment, University of Oulu, Finland. His main areas of interest are biosensingand separation science.