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Electrochimica Acta 133 (2014) 294–301

Contents lists available at ScienceDirect

Electrochimica Acta

j our na l ho me pa g e: www.elsev ier .com/ locate /e lec tac ta

ighly selective electrochemical sensor for ascorbic acid based on aovel hybrid graphene-copper phthalocyanine-polyanilineanocomposites

aithip Pakapongpan, Johannes Philipp Mensing, Ditsayut Phokharatkul, Tanom Lomas,disorn Tuantranont ∗

anoelectronics and MEMs Laboratory, National Electronics and Computer Technology Center, Pathumthani, Thailand

r t i c l e i n f o

rticle history:eceived 9 February 2014eceived in revised form 26 March 2014ccepted 29 March 2014vailable online 18 April 2014

eywords:rapheneopper phthalocyaninescorbic acidolyaniline

a b s t r a c t

In this work, we designed a novel electrochemical sensing platform based on graphene (Gr)/copper(II)phthalocyanine-tetrasulfonic acid tetrasodium salt (CuPc)/polyaniline(PANI) nanocomposites. The pre-pared composites were used to modify screen printed electrodes (SPE) for selective determination ofascorbic acid (AA) in presence of dopamine (DA) and uric acid (UA). Copper phthalocyanine was immo-bilized on graphene sheets by �-� interaction by electrolytical exfoliation and resulting CuPc/graphenewas embedded in a PANI matrix to prevent leakage of Gr/CuPc from electrodes. The Gr/CuPc/PANInanocomposites were characterized by scanning electron microscopy (SEM), UV-vis spectroscopy, cyclicvoltammetry (CV) and amperometry. The prepared modified electrode presents good electrocatalyticproperties, fast response time, high stability and reproducibility. The performance of the sensor exhib-ited a linear range from 5 × 10−7 to 1.2 × 10−5 M with low a limit of detection of 6.3 × 10−8 M (S/N = 3)

−1

lectrochemical sensor and the sensitivity of the sensor was found to be 24.46 �A mM . Moreover, the nanocomposites showexcellent selectivity and lower potential for the oxidation of ascorbic acid. The novel sensor successfullyapplied to determination of AA in real samples with satisfactory results. This can open up new oppor-tunities for fast, simple and selective detection of AA and provide a promising platform for sensor orbiosensor designs for AA detection.

© 2014 Elsevier Ltd. All rights reserved.

. Introduction

Vitamin C, also known as ascorbic acid (AA), is a water sol-ble vitamin, widely present in many biological systems, fruitsnd vegetables. AA is commonly used to supplement inadequateietary intake, as anti-oxidant and plays an important role in theuman metabolism as a free-radical scavenger, which may help torevent radical induced diseases such as cancer and Parkinson’sisease. Furthermore, deficiency of AA can cause scurvy disease.

t is administered in the treatment of many disorders, includinglzheimer’s disease, atherosclerosis, cancer, infertility and in somelinical manifestations of HIV infections [1]. The determination ofA in various natural and prepared foods, drugs, physiological flu-

ds, fruit juices, soft drinks and vegetables is of great importanceor biological and agro-industries. Hence, many methods have beeneported for the determination of AA, such as chemiluminescence

∗ Corresponding author. Tel.: +66 2 5646900 ext 2111.E-mail address: adisorn.tuantranont@nectec.or.th (A. Tuantranont).

ttp://dx.doi.org/10.1016/j.electacta.2014.03.167013-4686/© 2014 Elsevier Ltd. All rights reserved.

[2,3], spectrometry [4,5] and chromatography [6,7]. However, thesetechniques lack specificity and are prone to interferences by otherreducing agents in the sample. Since AA is an electroactive com-pound, electrochemical techniques for its detection have receivedconsiderable interest due to their high sensitivity, easy operationand low cost. A major problem for the electrochemical determi-nation of AA is interference from uric acid (UA) and dopamine(DA) because they usually coexist in real samples. Therefore, over-lapping oxidation of AA, UA and DA occurs since their oxidationpotentials are very similar at bare electrodes. In addition, the oxi-dation product of UA can catalyze the oxidation of AA which leadsto electrode fouling, poor selectivity and reproducibility. Thus, theselective detection of AA in the presence of UA is a major goal inthis research field. In order to improve the selectivity and sensi-tivity various materials such as organic redox mediators [8–12],nanoparticles [13,14], polymers [15], carbon nanotubes [16–19]

and graphene [20,21] have been used in the modification of elec-trodes for determination of AA.

Graphene (Gr) is a two-dimensional layer of sp2 bonded car-bon atoms closely packed into a honeycomb lattice. Due to its

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S. Pakapongpan et al. / Electro

nique properties such as high surface area, high electrical conduc-ivity, and strong mechanical strength, graphene has been widelymployed for various applications such as field-effect transistors22,23], gas sensors [24,25], electromechanical sensors-biosensor26–28], nanoelectronics [29], batteries [30,31], supercapacitors32] and hydrogen storage [33]. In addition, graphene can easily be

odified by making use of strong �-� bonding between its surface-orbitals and �-orbitals of many other planar aromatic molecules.herefore, varieties of hybrid materials between graphene andther materials such as polymers, metals, metal oxides and evenarbon nanotubes etc. with new functions are promising to besed in varieties of applications including electrochemical sensingpplications. Earlier research has shown that conducting polymerodified electrode-materials have many advantages for fabrica-

ion of electrochemical sensors for the detection of analytes due toheir good electrical properties and homogeneity in electrochemi-al deposition, strong adherence to electrode surfaces and chemicaltability of the films. Polyaniline (PANI) is one of the most widelytudied conducting polymers. It is highly conductive, exhibits goodnvironmental stability and can easily be prepared. PANI can act as

suitable matrix for immobilization of biomolecules and mediatoror redox and enzymatic reactions and it exhibits impressive signalmplification and antifouling properties [34–36]. These propertiesake PANI exceptionally suitable for the fabrication of sensors

r biosensors. In addition, the usefulness of PANI and grapheneomposites has been reported for many applications such as super-apacitors [37–39], fuel cells [40] and electrochemical sensors [41].t is well known that metallo-phthalocyanines have been used suc-essfully in preparing modified electrodes for sensors due to theiratalytic activity for a wide range of redox processes, chemical andhysical properties such as thermal stability, well-defined redoxctivity in sensing applications [42–44] and are well recognizedor their excellent electrocatalytic activity for many compounds45–49], which is dependent on the central metal atom. It is pos-ible for phthalocyanines to act as substitutes for enzymes, sincehey can act as the active center of an enzyme molecule with theame efficiency and selectivity [50,51].

In this paper, we propose the novel hybrid material ofraphene/copper(II) phthalocyanine (CuPc)/polyaniline with sim-le fabrication method for highly specific AA detection and a newpproach to the synthesis of Gr/CuPc/PANI nanocomposites mod-fied SPE by using electrolytic exfoliation method. CuPc acts asatalyst for the oxidation of AA whereas graphene increases thelectroactive surface area, enhances the immobilization of CuPcnd promotes the electron transfer between CuPc and electrode.he PANI matrix supports adhesion to the electrode surface andrevents CuPc from leaking out of the active electrode material.oreover, screen printed electrodes (SPE) offer high-quantity pro-

uction of extremely inexpensive and disposable electrochemicalensors [52]. This technology allows the mass production of repro-ucible, inexpensive, simple, easy to use, portable and mechanicalobust strips. The combination of CuPc, graphene with PANI couldmprove the conductivity, stability and the performance of elec-rochemical sensors for determination of AA. This nanocompositeould be used as a platform for biosensor and biocatalyst applica-ions.

. Experimental

.1. Reagents and materials

Graphite rods were purchased from Electron Microscopyciences (Hatfield, Pennsylvania). Ascorbic acid, copper(II)hthalocyanine-tetrasulfonic acid tetrasodium salt and ani-

ine were purchased from Sigma Aldrich and used without further

ca Acta 133 (2014) 294–301 295

purification. 0.1 M phosphate buffer saline (PBS, pH 7.0) was pre-pared by mixing solutions of Na2HPO4 and NaH2PO4. All solutionswere prepared with deionized water.

2.2. Apparatus

All electrochemical experiments were performed with a com-puter controlled �-Autolab modular electrochemical system (EcoChemie Utrecht, The Netherlands). This instrument was operatedusing the GPES program (Eco Chemie) in a conventional three elec-trode electrochemical cell using screen printed carbon electrode asworking electrode, platinum wire as counter electrode and Ag/AgClin saturated KCl as the reference electrode.

2.3. Preparation of Gr/CuPc/PANI composite material modifiedelectrode

Gr/CuPc has been prepared by the electrolytic exfoliation ofgraphite anodes in electrolyte containing CuPc as previouslyreported by our group [53]. 5 mg ml−1 CuPc were dissolved indeionized water and electrolysis was performed with a con-stant potential of 17.5 V at room temperature for approximately15 hours. The potential forces the CuPc anions towards the sur-face of the graphite anode. This result in the successive exfoliationof the anode material into single and few layer graphene sheetswhich are stabilized by �-� bonded water soluble CuPc anions.The exfoliation process is followed by ultrasonication for 1 hour tofurther exfoliate larger graphite particles into few-layer graphenesheets. The obtained suspension was then filtered with filter paper.Subsequently, remaining large particles were removed by centrifu-gation at 5000 rpm for 30 minutes and discarding of the sediment.This suspension of CuPc stabilized graphene was subsequently usedfor fabrication of Gr/CuPc/PANI composite. Hydrochloric acid (37%)was added to the suspension to obtain an HCl concentration of 1 M.Then aniline was added to the acidic suspension in a concentra-tion of 0.01 M. Afterwards, the mixture was ultrasonicated again for30 minutes in order support formation of �-� stacks of graphenesheets and aniline molecules. To initiate the polymerization of PANIthe suspension was rapidly mixed with an equal volume of a 2.5 mMsolution of ammonium persulfate (APS) in 1 M hydrochloric acidand left stirring overnight. After the polymerization the solid prod-uct is separated from the liquid phase by vacuum filtration with a0.22 �m pore diameter nitrocellulose membrane and washed withethanol and DI water. Finally, the composite product was collectedand dispersed in 200 �l DI water. The Gr/CuPc/PANI nanocompos-ite electrodes were fabricated by casting 5 �l of the solution on thesurface of a screen printed electrodes and followed by air dryingfor 8 hours (shown in Scheme 1).

3. Results and Discussion

3.1. Characterization of Gr/CuPc/PANI modified electrode

After the preparation, the morphology of Gr/CuPc/PANInanocomposites was characterized by scanning electronmicroscopy (SEM). Fig. 1A and1B show the typical layered sheetsstructure of graphene with a rippled morphology of the flake-likeshapes randomly packed in stacked structures which demon-strated that graphene sheet structure was obtained. The strongattractive forces between graphene sheets and CuPc molecules,due to �-� interaction, lead to composite formation. However,the adsorption of CuPc on the Gr surface cannot be seen clearly

in SEM images. Aniline was polymerized onto Gr/CuPc, forminga film of homogenous surface morphology. After polymerizationof PANI, as shown in Fig. 1 C and 1D, it can be seen that a film ofGr/CuPc/PANI with a layered and wrinkled structure resides on

296 S. Pakapongpan et al. / Electrochimica Acta 133 (2014) 294–301

Scheme 1. The Illustration of the preparation process of Gr/CuPc/PANI nanocomposites.

and (C

trPGovm

Ft∼oCtsowlbsoee

which suggest molecular interaction between the constituents ofthe nanocomposite. The results confirm that CuPc absorbed ongraphene sheet and the nanocomposites were successfully pre-pared.

Fig. 1. SEM images of (A, B) Gr/CuPc

he surface of the graphene sheets. The layered structures appearougher than the morphology of pure Gr/CuPc, indicating thatANI has been successfully combined onto the surface of Gr/CuPc.r/CuPc/PANI nanocomposite proved beneficial for the fabricationf sensor platforms due to the graphene structure which pro-ides a large surface area for further immobilization of variousolecules.The UV-vis spectra of the investigated materials are shown in

ig. 2 (a-d). The spectrum of CuPc (Fig. 2a) shows the typical fea-ures of metal phthalocyanines. Absorption peaks at ∼610 nm and690 nm correspond to � → �* transitions whereas the shoulderf the spectrum in the blue region is due to n → �* transitions [53].urve b depicts the spectrum of CuPc/PANI. The spectrum containshe previously described CuPc-features with PANI absorption peaksuperimposed. The peak at ∼330 nm is due to � → �* transitionsf PANI whereas an n → �* transition band is located at ∼610 nmhich increases the absorbance of the peak centered at this wave-

ength [54]. The remaining spectra (c, d) show CuPc and CuPc/PANIoth combined with graphene. As can be clearly observed inclu-ion of graphene does not alter relative intensities and positions

f the peaks at ∼610 nm and ∼690 nm since graphene does notxhibited any absorption peaks in measured spectral range. How-ver, the PANI � → �* peak in curve d is red-shifted to ∼340 nm

, D) Gr/CuPc-PANI nanocomposites.

Fig. 2. UV–vis absorption spectra of CuPc (curve a), CuPc/PANI (curve b), Gr/CuPc(curve c) and Gr/CuPc/PANI (curve d).

S. Pakapongpan et al. / Electrochimica Acta 133 (2014) 294–301 297

Fig. 3. CVs of (a) bare SPE, (b) CuPc/PANI/SPE and (c) Gr/CuPc/PANI/SPE, respectivelyi

3e

i(r(r(sw0CppgcCtGitofiocaieaeta

GTiswicaaot

potential (E0 ) of Gr/CuPc/PANI nanocomposites has changed linear

n 0.1 M PBS pH 7.0 at scan rate 100 mV/s. Inset: the stability of the nanocomposites.

.2. Electrochemical properties of Gr/CuPc/PANI modifiedlectrode

The electrochemical properties of nanocomposites were stud-ed using cyclic voltammetry. Fig. 3 shows cyclic voltammogramsCVs) of different modified electrodes in 0.1 M PBS pH 7.0 at a scanate of 100 mV/s. There was no redox peak observed at bare SPEcurve a), CuPc/PANI (curve b) showed a small background cur-ent with a weak redox peak of CuPc and anodic (Epa) and cathodicEpc) peak potentials at 0.11 V and −0.17 V, respectively. It can beeen, in curve c, that a pair of redox peaks at 0.067 V and −0.09 Vere clearly observed with a peak to peak separation (�Ep) of

.157 V and formal potential (E0′) of −0.012 V which indicates that

uPc was successfully immobilized on the surface of graphene. Therocess of the Cu(II)/Cu(III) redox reaction in the central cavity ofhthalocyanine explains the peaks observed above. By combiningraphene and CuPc, electron transfer between CuPc and electrodean be enhanced. It also increases greatly the adsorbed amount ofuPc due to the high surface area of graphene and also increaseshe conductivity of the material. However, the immobilization ofr/CuPc on the electrode surface is limited. For this reason, PANI

s included serving as a matrix for Gr/CuPc to support immobiliza-ion on the electrode surface. This effectively prevents diffusion outf the polymer matrix film. The stability of nanocomposite modi-ed electrode was investigated as shown in the inset of Fig. 3. Nobvious changes after scanning for 100 cycles were observed. Theurrent response of Gr/CuPc/PANI modified SPE showed that anodicnd cathodic peak currents were stable and almost retained thenitial response. It was found that the nanocomposites modifiedlectrode had an excellent stability due to the strong �-� inter-ction between CuPc and graphene sheets. Immobilization on thelectrode surface could be improved by using a PANI matrix. Addi-ionally, the ratio of Ipa/Ipc was approximately equal to 1, suggesting

fast and quasi-reversible electron transfer process.The effect of scan rate on the electrochemical response of

r/CuPc/PANI modified SPE was investigated as shown in Fig. 4.he oxidation and reduction peak currents increased linearly withncreasing scan rate from 10 to 100 V/s (Fig. 4A).The linear regres-ion equations were y = 0.72x + 6.763 and y = −0.766x − 10.911ith correlation coefficients of 0.998 and 0.991. These results

ndicate that the nanocomposites enabled reversible surface-ontrolled electrochemical reaction processes. The plots of anodicnd cathodic peak potentials versus logarithm of the scan rate show

linear relationship (inset B of Fig. 4) with a correlation coefficientf 0.991 and 0.998. The plots of the anodic and cathodic peak poten-ials vs. the logarithm of the scan rates yielded two straight lines

Fig. 4. CV of Gr/CuPc/PANI modified electrode in 0.1 M PBS pH 7.0 at a scan rate of10-100 mV/s (from inner to outer). Insets: (A) the plot of peak currents vs. scan ratesand (B) the plot of peak potential vs. log v.

with slopes of 2.3RT/(1 − ˛)nF and −2.3RT/˛nF, respectively. Fromthe slopes and the Laviron theory, Eq. (1-3),

Epc = E0′ − 2.3RT

˛nFlog v (1)

Epa = E0′ + 2.3RT

(1 − ˛)nFlog v (2)

log ks = ̨ log(1 − ˛) + (1 − ˛) log ̨ − logRT

nFv− ˛(1 − ˛)nFEP

2.3RT(3)

where v is the scan rate, R is the gas constant, T is the temper-ature, n is the number of electron transfer and F is the Faraday’sconstant. From these equations it follows that the charge transfercoefficient (�) and the heterogeneous electron transfer rate con-stant (ks) were estimated to be 0.51 and 1.4 s−1, respectively [55].The number of electron transfers involved in the electrochemi-cal mechanism was assumed as 1. This result suggested that thenanocomposites facilitate the fast electron transfer with high effi-ciency kinetic process. According to Eq. (4),

Ip = n2F2vA�c

4RT= nFQv

4RT(4)

where Ip is the peak current, Q is the charge obtained by inte-grating the cathodic peak at low voltage scan rate, A is the areaof the working electrode and � is the surface coverage (mol/cm2).The � of CuPc on graphene modified electrode was so calculated tobe 8.8 × 10−9 mol/cm2. Which is approximately 62.4 times higherthan the � value of CuPc/PANI (� = 1.41 × 10−10 mol/cm2), sug-gesting that the composite with graphene has higher CuPc loadingdue to graphene provide a large surface area for CuPc immobiliza-tion.

3.3. Effect of solution pH on the Gr/CuPc/PANI modified electrode

The influence of the pH value on the electrochemical behaviorof Gr/CuPc/PANI nanocomposites was investigated. The CVs of thenanocomposites modified electrode in Fig. 5 show a dependence onthe pH conditions. The anodic and cathodic peak potentials shiftedto negative potentials with increased pH from 5.0 to 9.0 at scanrate of 100 mV/s. Additionally, the CV at pH 5.0 revealed two sep-arate oxidation and reduction peaks which are typically found forPANI since PANI acts as mediator in acidic conditions. The formal

′

relationship with pH of the solution. As shown in inset Fig. 5, theplot of formal potential and pH value show linearity with the slopeof −71 mV pH−1 (r2 = 0.999). This value was close to the theoretical

298 S. Pakapongpan et al. / Electrochimica Acta 133 (2014) 294–301

Fp

viir

3

tIcttiIceTiAp

C

2

3

t

F5

Fig. 7. (A) Amperometric current response of Gr/CuPc/PANI modified SPE to suc-

ig. 5. CVs of Gr/CuPc/PANI/SPE in 0.1 M PBS with pH 5-9, Inset: The plots of formalotential vs. pH values at a scan rate: 0.1 V s−1.

alue of −59 mV pH−1 for a reversible redox reaction and suggest-ng that the numbers of electron and proton transferred involvedn the electrochemical reaction process equal the electrochemicaleaction expressed in Eq. (5).

.4. Electrocatalytic oxidation of AA at the modified electrode

The electrocatalytic activity of Gr/CuPC/PANI modified SPEowards AA was investigated and results are shown in In Fig. 6.n the absence of AA (curve a), no oxidation and reduction peakurrent change was observed. After addition of different concen-rations of AA from 50 �M to 0.5 mM, as shown in curve b to e,he oxidation peak current increases proportional with increas-ng AA concentration and the reduction peak current decreases.t demonstrated that the current response increase due to CuPcan catalyst the oxidation of AA and the Gr/CuPc/PANI modifiedlectrode has good electrocatalytic activity toward AA oxidation.he electrocatalytic mechanism of AA to the nanocomposites mod-fied electrode can be described by the Eq. (5), (6). CuPc(III) oxidizesA to dehydroascorbic acid (DHAA) by the Cu ion at the central ofhthalocyanine structure and regenerates CuPc(II).

uPc(II) ↔ CuPc(III) + e− + H+ (5)

CuPc(III) + AA → 2CuPc(II) + DHAA + 2H+ (6)

.5. Amperometric response of the sensing to AA

The determination of AA concentration was performed usinghe amperometric method. In order to get a high current response

ig. 6. CVs of Gr/CuPc/PANI/SPE in 0.1 M PBS pH 7.0 without AA(a) and with0 �M(b), 0.1 mM(c), 0.25 mM(d) and 0.5 mM(e) AA at a scan rate of 20 mV/s.

cessive addition of different AA concentration. Inset: The calibration curve of the

currents vs. concentration of AA from 0.5 to 18 �M. (B) Amperometric responseof Gr/CuPc/PANI modified SPE to 2 �M AA, 0.1 mM UA, 0.1 mM DA and 2 �M AA,respectively in 0.1 M PBS pH 7.0 at applied potential of +0.1 V vs. Ag/AgCl.

and avoid interference, the examination of the effect of appliedpotential on the sensor was studied in 0.1 M PBS pH 7.0 at differ-ent potentials ranging from 0 V to +0.6 V versus Ag/AgCl (Fig. S1).The current response increased with increased potential from 0 V to+0.3 V vs. Ag/AgCl and became stable at +0.1 V vs. Ag/AgCl. The high-est current response was measured at +0.2 V vs. Ag/AgCl. However,at potentials higher than +0.1 V vs. Ag/AgCl no significant currentincrease was observed. Thus, +0.1 V vs. Ag/AgCl was selected asoptimal potential due to the sensor still maintaining high sensitiv-ity and diminishing possible interference from other electroactivespices at high potentials. The amperometric current response ofthe Gr/CuPc/PANI modified SPE to successive additions of differentconcentrations of AA was evaluated under optimum conditions asshown in Fig. 7. The applied potential was fixed at +0.1 V vs. Ag/AgCl.After addition of AA, the oxidation current response increased with

increased concentration of AA. The sensor reached 95% of steadystate within 4 s, indicating a fast amperometric response to AA. Themeasured peak current response showed proportional relation toAA concentration in the range from 5 × 10−7 M to 1.8 × 10−5 M. The

S. Pakapongpan et al. / Electrochimica Acta 133 (2014) 294–301 299

Table 1Comparison of the performance of the proposed sensor with some modified electrodes used for the determination of ascorbic acid.

Modified electrodes Potentials (V) Linear range (�mol l−1) LOD (�mol l−1) Sensitivity (�A/mmol l−1) Ref.

FCAM/CPE +0.248 34.8 - 490 10.8 - [9]NanoCoPc/GCE - 0.55 - 55000 0.1 - [56]Poly(direct blue 71)/GCE +0.1 1 - 10000 1 - [57]Graphene doped CPE +0.31 0.1 - 106 0.07 3.31571 [21]CoTNPPc-MWNTs/GCE +0.215 10 - 1600 5 - [58]CoPc-MWNTs/GCE +0.19 10 - 2600 1 - [59]BPEI-EGDE/[Fe(CN)6]4/GCE +0.24 1 - 5000 1 28.2 [60]Gr/CuPc/PANI/SPE +0.1 0.5 - 120 0.063 24.46 This work

FCAM, ferrocene carboxylic acid; CoTNPPc, cobalt phthalocyanine nanoparticles; MWNTs, multi-walled carbon nanotubes; CPE, carbon paste electrode; BPEI, Branchedpolyethylene mine; EGDE, Ethylene glycol diglycidyl ether; [Fe(CN)6]4, Hexacyanoferrate

Table 2Determination of AA in real samples (n = 3).

Sample Content (�mol l−1) Add (�mol l−1) Found (�mol l−1) Recovery (%) RSD (%)

cllanToTat

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tGr(owdtppttlCtAc

3

wtoumpata7

1 8.34 10

2 16.5 10

3 13.1 10

alibration curve of the sensor is shown in the inset of Fig. 7A. Theinear range of the sensor was 5 × 10−7 to 1.2 × 10−5 M with theinear equation y = 24.46x + 2.125, a correlation coefficient of 0.998nd a limit of detection estimated to be 6.3 × 10−8 M at signal tooise ratio of 3(S/N = 3) with high sensitivity of 24.46 �A mM−1.he comparison of the performance of Gr/CuPc/PANI with previ-us works of some mediator based AA sensors was summarized inable 1. It can be seen that this sensor exhibited higher sensitivitynd lower detection limit than most of other sensors reported dueo large surface area and good conductivity of graphene.

.6. Repeatability and stability of the Gr/CuPc/PANI/SPE

The reproducibility and stability of the sensor were also inves-igated by the amperometric method. The reproducibility of ther/CuPc/PANI modified electrode was evaluated from the current

esponse at +0.1 V vs. Ag/AgCl and the relative standard deviationRSD) value of 3.03% was observed for 10 successive measurementsf 2 �M AA. The fabrication reproducibility was also determinedith AA at five different modified electrodes prepared indepen-ently and 5.1% of RSD were achieved. The results demonstratehat the reproducibility of the sensor was acceptable and had goodrecision. The long term stability of the Gr/CuPc/PANI nanocom-osites modified SPE was studied by storing the electrode at roomemperature for 3 weeks in 0.1 M PBS pH 7.0. After this time periodhe sensor retained 92.5% of its initial response, indicating goodong term stability due to the strong interaction of graphene withuPc and PANI film, preventing CuPc from leaking out of the elec-rode surface Which is a requirement for this type of sensors forA determination in in-vivo monitoring applications such as theirculatory system of the human body.

.7. Interference study

It is well known that uric acid (UA) and dopamine (DA) coexistith AA in biological samples. This is a major problem in the detec-

ion of AA, since they are oxidized at almost the same potential andccur at high potential at bare electrodes. Cyclic voltammetry wasse to examined the oxidation of UA and DA at the nanocompositesodified electrode. When UA and DA are presence, the oxidation

eak potentials occur at +0.5 V and +0.64 V, respectively without

ny catalytic reaction with CuPc as shown in Fig.S2. Fig. 7B showshe amperometric current response of Gr/CuPc/PANI to successivedditions of 2 �M AA, 0.1 mM UA and 0.1 mM DA in 0.1 M PBS pH.0 at the applied potential of +0.1 V vs. Ag/AgCl. It can be seen that

18.25 99.1 2.5728.1 116 4.3323.82 107.2 2.98

no current response increase was observed on the nanocompos-ites modified SPE after addition of UA and DA. This indicates thatpresence of UA and DA did not interfere with AA peak current andthe nanocomposites have high selectivity in presence of high coex-isting substances. This sensor could be applied to determine AA inthe real samples.

3.8. Real sample analysis

In order to develop an analysis application tool, theGr/CuPc/PANI nanocomposite modified electrode for determi-nation in real samples was investigated by analysis of AA incommercial vitamin C tablets as shown in Table 2. Vitamin Ctablets were dissolved in 0.1 M PBS pH 7.0 and a standard additionmethod was applied by addition of a known concentration of AAinto the test solution. The recovery for the determination of AA wasin the range of 99.1–116% for three samples. The recovery and RSDwere satisfactory, indicating the good accuracy of the proposedsensor. Hence, the proposed sensor was suitable for determinationof AA in real samples.

4. Conclusions

We have successfully prepared Gr/CuPc/PANI nanocompositesmodified SPE by electrolytic exfoliation method and applied it forconstructing an AA sensor. Graphene provides a large surface areaand strong interaction with adsorbed CuPc. PANI was not onlyused as an immobilization matrix but also helped to increase con-ductivity. The nanocomposite shows an excellent electrocatalyticactivity toward AA, high selectivity, sensitivity, wide range and lowdetection limit. The results indicate that the graphene and PANIcan promote the electron transfer of AA at the electrode and alsoimprove the conductivity and stability. Moreover, the sensor hasbeen applied to determination of AA in real samples with sat-isfactory results. This sensor platform combines easy fabricationand high selectivity for AA, which has great potential for sensoror biosensor applications of several analytes in clinical diagnosis,pharmaceutical analysis and in the field of bio-electrochemistry.

Acknowledgments

This work was supported financially by National Scienceand Technology Development Agency (NSTDA) and NECTEC. A.

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ppendix A. Supplementary data

Supplementary data associated with this article can be found,n the online version, at http://dx.doi.org/10.1016/j.electacta.014.03.167.

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