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View Article OnlineView Journal | View Issue

aSchool of Chemical and Biomedical Engine

62 Nanyang Drive, Singapore, 637459sbCentre for Nanobers and Nanotechnol

Singapore 117576. E-mail: seeram@nus.ed

6593cDepartment of Mechanical Engineering, Na

117576dInstitute of Physical Chemistry, Polish Acad

224 Warszawa, Poland. E-mail: ktpkannan

+48-223 433 375. Fax: +48-223 433 333

† Electronic supplementary informa10.1039/c3an00070b

‡ Both authors contributed equally to this

Cite this: Analyst, 2013, 138, 1779

Received 24th November 2012Accepted 17th January 2013

DOI: 10.1039/c3an00070b

www.rsc.org/analyst

This journal is ª The Royal Society of

Nanostructured a-Fe2O3 platform for theelectrochemical sensing of folic acid†

Thandavarayan Maiyalagan,‡a J. Sundaramurthy,‡bc P. Suresh Kumar,bc

Palanisamy Kannan,*d Marcin Opallo*d and Seeram Ramakrishna*bc

a-Fe2O3 nanofibers are synthesized by a simple and efficient electrospinning method and the selective

determination of folic acid (FA) is demonstrated in the presence of an important physiological

interferent, ascorbic acid (AA), using the a-Fe2O3 nanofiber modified glassy carbon (GC) electrode at

physiological pH. Bare GC electrode fails to determine the concentration of FA in the presence of a

higher concentration of AA due to the surface fouling caused by the oxidized products of AA and FA.

However, modification with a-Fe2O3 nanofibers not only separates the voltammetric signals of AA and

FA by 420 mV between AA and FA, but also enhances higher oxidation current. The amperometric

current response is linearly dependent on FA concentration in the range of 60–60 000 nM, and the

a-Fe2O3 nanofiber modified electrode displayed an excellent sensitivity for FA detection with an

experimental detection limit of 60 nM (1.12 � 10�10 M (S/N ¼ 3)). Furthermore, the a-Fe2O3 nanofiber

modified electrode showed an admirable selectivity towards the determination of FA even in the

presence of a 1000-fold excess of AA and other common interferents. This modified electrode has been

successfully applied for determination of FA in human blood serum samples.

1 Introduction

In recent years, the synthesis and fabrication of nanomaterialswith tailoring their size, morphology, and porosity have beenintensively pursued not only for fundamental scientic interestbut also for many technological applications.1–3 Nanoparticles(zero-dimensional (0-D)) and nanowires/nanorods (one-dimen-sional (1-D)) with controlled size and shape are of key importancebecause their electrical, optical, and magnetic properties arestrongly dependent on their size and shape.1–3 Currently, one-dimensional (1-D) nanomaterials such as silicon nanowires(SiNWs), carbon nanotubes (CNTs), and conducting polymernanowires (CP NWs) have opened the possibility to fabricateelectrochemical sensors and biosensors.4–7 Their high sensitivityand new sensing mechanisms are related to intrinsic propertiesassociated with a high surface-to-volume ratio.4–7 Further, 1-D

ering, Nanyang Technological University,

ogy, National University of Singapore,

u.sg; Fax: +65-6872 5563; Tel: +65-6516

tional University of Singapore, Singapore

emy of Sciences, 44/52 ul. Kasprzaka, 01-

@gmail.com; mopallo@ichf.edu.pl; Tel:

tion (ESI) available. See DOI:

work.

Chemistry 2013

nanostructures can be used for both efficient transport of elec-trons and optical excitation, and these two factors make themcritical to the function and integration of nanoscale devices, andhave been the focus of intensive research for many potentialapplications in electronics, photonics, drug delivery, medicaldiagnostics, and magnetic materials.8–11

Hematite (a-Fe2O3) is the most stable iron oxide with n-typesemiconducting properties (Eg ¼ 2.2 eV) under ambient condi-tions. It has been intensively investigated because of its wideapplications in catalysts, pigments, magnetic materials, gassensors, and lithium ion batteries.4,12–15 Fe2O3 was generallyconsidered to be biologically and electrochemically inert, and itselectrocatalytic functionality has been rarely realized directly inthe past,16 whereas Fe2+ ions (instead of Fe3+) play the dominantrole in the oxidation reaction.17–19 Meanwhile, Fe2O3 was alsodemonstrated to show both reversible reduction and reversibleoxidation of Fe(III) in a basic carbonate buffer solution.17 Never-theless, in contrast with interests focusing on synthetic andcatalytic applications of Fe3O4, reports on the electrochemicalcharacterization of Fe2O3 nanoparticles are rather rare, and littleattention has been paid to the detailed study of their sensingperformance.20–22 In principle, Fe2O3 nanoparticles may effi-ciently mediate the nal heterogeneous chemical oxidation orreduction of the target agent, while the converted iron oxides canbe continuously and simultaneously recovered by electro-chemical oxidation or reduction due to their high surface tovolume ratio. From this key point, an electrocatalytic study ofnanostructured Fe2O3 in biocompatible environments may not

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only be of scientic interest, but could also produce real benetssuch as their substitution for noble metals/enzymes for practicalenzyme-free biosensor applications. Due to its low cost, goodstability, and reversibility, a-Fe2O3 has been proven to be animportant semiconductor nanomaterial for electrochemicalsensors.23 However, so far, there have been only a few reports onbio-sensing properties of 1-D nanostructural a-Fe2O3. Generally,the properties of a biosensor are strongly dependent on itssurface area. The relatively low surface to volume ratio ofconventional bulk a-Fe2O3 materials leads to their poor bio-sensing properties. Hence, developing 1-D nanostructure a-Fe2O3

with a high surface area is very important for increasing theirapplications in sensors.4,14 Recently, as prepared a-Fe2O3 nano-material has been proven to be a successful electrode materialdue to the intrinsic peroxidase-like catalytic activity.24 Thea-Fe2O3 nanowire array modied glucose sensor exhibited anexcellent biocatalytic performance towards the oxidation ofglucose with a detection limit of 6 mM (S/N ¼ 3).24 In the presentstudy, we have synthesized a-Fe2O3 nanober by a simple elec-trospinning method and used it to improve the bio-sensingperformance towards the oxidation of FA for the rst time.

Folic acid, known as a widely used water soluble vitamin, isreported to be a very signicant component for human healthwhich relates to a series of diseases such as gigantocytic anemia,leucopoenia, mental devolution, heart attack and congenitalmalformation.25–28 FA is one of the important coenzymes of thehaematopoietic system that controls the generation of ferro-haeme.28 The dosage of FA is associated with the treatments ofhyperhomocysteinemic coronary artery disease, hypertension,depression, hypercholesterolemia, mammary tumor, vasculardisease and neural tube defects of pregnant women.29–33 Theimportant biomolecules such as AA and FA are present in humanblood plasma,34,35 urine36,37 and blood serum samples.38,39 Sincethese biomolecules coexist in human uids, their simultaneousdetermination is essential to secure the human health from theabove critical diseases risk. Therefore, the selective and sensitivedetermination of FA is very important from the clinical andhealth viewpoints. In this paper, we will show that the a-Fe2O3

nanober modied GC electrode exhibits an excellent electro-catalytic activity towards FA, and a detection limit of 60 nM FAhas been achieved using the amperometry method. The a-Fe2O3

nanober array on the GC electrode with a nano-size and coarsesurface provides a platform for FA oxidation by contributing bothexcess electroactive sites and strong adhesion to the GC electrodesurface, which results in the enhanced sensitivity and long termstability of the a-Fe2O3 electrode. The application of the Fe2O3

modied electrode has been successfully demonstrated bymeasuring the concentration of FA in real samples.

2 Experimental section2.1 Materials and methods

Polyvinylpyrrolidone (PVP; MW ¼ 1 40 000) and iron(III) acety-lacetonate (Fe(acac)3) were purchased from Sigma-Aldrichand Fluka, Singapore, respectively. Ethanol (HPLC grade) andglacial acetic acid were purchased from Tedia, Singapore andused as received. The biomolecules, uric acid (UA) and folic acid

1780 | Analyst, 2013, 138, 1779–1786

(FA), were purchased from Merck chemicals and were used asreceived. All other chemicals used in this investigation were ofanalytical grade. The phosphate buffer solution (PBS; pH ¼ 7.2)was prepared using Na2HPO4 and NaH2PO4. Double distilledwater was used to prepare the solutions in this investigation.

2.2 Preparation of a-Fe2O3 nanobers

Firstly, 1 g of PVP was dissolved in 10 mL of ethanol solutionand homogeneously stirred at room temperature for 1 h forcomplete dissolution. Then, 0.4 g of Fe(acac)3 precursor wasadded to the PVP solution and continuously stirred for 6 hfollowed by addition of 1 mL acetic acid. Finally, 5 mL ofFe(acac)3–PVP precursor solutions were loaded in a 5 mL plasticsyringe with a hypodermic needle (dia. 27 G). The hypodermicneedle was then connected to a high-voltage supply capable ofgenerating direct current (DC) voltages up to 30 kV. Electro-spinning was carried out by applying a power supply of around16.5 kV at the needle in a controlled electrospinning set-up(Electrospunra, Singapore). Aluminum foil was used as thecounter electrode, and the distance between the needle and thecollector was maintained at 15 cm. The as-spun Fe(acac)3–PVPcomposite nanober mats were placed in an advanced vacuumoven at room temperature for 12 h to remove the solventresiduals. Finally, the nanobers were calcined at 500 �C for 5 hin air at a heating rate of 5 �C min�1, and nally a-Fe2O3

nanobers were obtained and stored carefully.

2.3 Instrumentation

The crystallographic information of the prepared a-Fe2O3

nanobers was studied using the powder X-ray diffractiontechnique (XRD, Shimadzu XRD-6000, Cu Ka radiation oper-ating at 30 kV/40 mA). The surface morphologies of the nano-structures were characterized using a eld emission-scanningelectron microscope (FE-SEM) JEOL JSM-6301F. Transmissionelectron microscopy (TEM), JEM-2010, JEOL USA Inc., wasemployed to study the surface morphology of a-Fe2O3 nano-bers. The electron beam accelerating voltage of themicroscopewas at 200 kV. Electrochemical measurements were performedin a conventional two compartment three electrode cell with amirror polished 3 mm glassy carbon (GC) as the working elec-trode, Pt wire as the counter electrode and a NaCl saturatedAg/AgCl as the reference electrode. The electrochemicalmeasurements were carried out with a CHI Model 660C (Austin,TX, USA) electrochemical workstation. In cyclic voltammetry,the electrochemical oxidations of AA and FA were carried out ata scan rate 50 mV s�1. Pulse width ¼ 0.06 s, amplitude¼ 0.05 V,sample period ¼ 0.02 s and pulse period ¼ 0.20 s were used indifferential pulse voltammetry (DPV). For chronoamperometricmeasurements, pulse width ¼ 0.25 s and potential step ¼ 1 mVwere used. All the electrochemical measurements were carriedout under a nitrogen atmosphere at room temperature (27 �C).

2.4 a-Fe2O3 nanober modied electrodes

The a-Fe2O3 nanostructure modied GC electrode was preparedas follows. First, the surface of the glassy carbon electrodefor each experiment was mechanically polished with 600 grit

This journal is ª The Royal Society of Chemistry 2013

Fig. 2 XRD pattern of Fe(acac)3–PVP nanofibers calcined at 500 �C for 5 h at aheating rate of 5 �C min�1 in air.

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sand-paper and 0.050 mm alumina powders, which was thenrinsed with acetone and double distilled water. A 3 mL aliquot ofa-Fe2O3 nanobers (dispersed in water, 5 mg mL�1, and pH ¼7.0) was dropped onto the surface and dried under atmosphericconditions. In addition, a bulk-Fe2O3 electrode (denoted asb-Fe2O3 GC electrode, fabricated by the above mentionedprocess) was used for comparison.

3 Results and discussion3.1 Characterization of the as-prepared a-Fe2O3

nanostructure

The morphology of as-electrospun Fe(acac)3–PVP compositenanober before and aer calcination was investigated by per-forming FESEM analysis. Fig. 1a shows the formation of ahighly interconnected network of nanobers with an averageber diameter of 288 nm. Fig. 1a (inset) shows the distributionof bers upon applying a potential of 16.5 kV; the broaddistribution of the bers was due to the dominancy ofCoulombic repulsive forces upon applying such a higherpotential. The formation of nanobers was observed aercalcination of the Fe(acac)3–PVP composite at 500 �C for 5 h at arate of 5 �C min�1 in air (Fig. 1b). The novel morphology ofnanostructures with ellipsoidal shape of a-Fe2O3 nanoparticlesuniformly plaited along the ber directions was observed. Thismorphology was due to the combined effect of phase separation(thermodynamic) and electrospinning (electro-hydrody-namic).40 The phase separation of the polymer and theprecursor induced the formation of precursor islands, and theelectrospinning coerced the precursors to plait together withthe result of spinning and whirling effects upon applyingpotential. During calcining, the polymer PVP present all overthe brous structure was decomposed and yielded nanorod-likestructures. The nanobers have a coarse surface due to theadsorption and assembly of small crystalline nanoparticles(Fig. 2b; inset), some of which even have a chain-likemorphology. In comparison with the randomly packed particlecounterpart, such arrayed nanowires provide more orderedspatial orientation and improved structural stability. As a result,higher mass transfer and permeation rate, a stable porousvolume and less structural corruption can be expected duringthe electrochemical recycling. In addition, the coarse surface

Fig. 1 (a) FE-SEM image of as-electrospun Fe(acac)3–PVP composite nanofibersat a power supply of 16.5 kV (inset: histogram of the nanofiber diameter anddistribution) and (b) FE-SEM image of a-Fe2O3 nanofibers after calciningcomposites at 500 �C for 5 h at a ramp rate of 5 �C min�1.

This journal is ª The Royal Society of Chemistry 2013

may also result in enhanced long-term stability due to a moresecure attachment to the electrode surface.

Further, the XRD analysis on calcined nanobers has beencarried out to conrm the a-Fe2O3 phase formation. Fig. 2shows the XRD pattern of a-Fe2O3 nanobers aer calcinationof the Fe(acac)3–PVP composite at 500 �C for 5 h in air. All thediffraction peaks were well indexed to the rhombohedralhexagonal phase of hematite (a-Fe2O3) (JCPDS: 33-0664). Thestrong and narrow-sharp diffraction peaks showed the purityand high degree of crystallization of synthesized a-Fe2O3

nanobers.

3.2 Electrochemical oxidation of FA

We have examined the electrocatalytic activity of a-Fe2O3 GC, b-Fe2O3 GC and unmodied GC electrodes towards the oxidation ofFA. We found that the a-Fe2O3 nanober modied GC electrodeshowed higher electrocatalytic activity towards AA and FA thanthe b-Fe2O3 GC and unmodied GC electrodes. Fig. 3A shows thecyclic voltammograms (CVs) obtained for 0.25mMFA at bare anda-Fe2O3 nanober modied GC electrodes in a 0.20 M phosphate

Fig. 3 (A) CVs obtained for 0.25 mM FA at bare and a-Fe2O3 nanofiber modifiedGC electrodes after the 1st (a and d), 10th (b and e) and 20th (c and f) cycles in a0.2 M PB solution at a scan rate of 50 mV s�1 and (g) CV obtained for the a-Fe2O3

nanofiber modified GC electrode in the absence of 0.5 mM FA in a 0.2 M PBsolution. Inset: bulk-Fe2O3 modified GC electrode in the presence of 0.25 mM FAin 0.2 M PB solution. (B) Anodic peak current vs. square root of scan rates.

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buffer (PB) solution (pH ¼ 7.2). At the bare GC electrode, anoxidation peak was observed for FA at 0.95 V in the rst cycle(curve a). In the subsequent cycles, the FA oxidation peak wasshied to more positive potential with decreased peak current.Aer 20 cycles, the oxidation peak of FA almost disappeared(curve c), indicating that the bare GC electrode was not suitablefor the stable and simultaneous determination of FA. Theadsorption of the oxidized product of FA on the electrode surfaceis the possible reason for the decreased FA oxidation current andmore positive peak shi in the oxidation potential at the bare GCelectrode. On the other hand, a well-dened oxidation peak wasobserved at 0.81 V for FA at the a-Fe2O3 nanober modied GCelectrode (curve d), which was a 140 mV less positive potentialthan at the bare GC electrode. It can be seen from Fig. 3A that theoxidation potential of FA remained stable even aer 20 repeatedpotential cycles (curve f), indicating that the oxidation of FA washighly stable at the a-Fe2O3 nanober modied GC electrode.The a-Fe2O3 nanober modied GC electrode did not show anyoxidation response in the absence of FA (curve g). These resultsindicated that the a-Fe2O3 nanobers are excellent candidatestoward the electrochemical oxidation of FA.

For comparison, we have also modied the electrode withbulk Fe2O3 (b-Fe2O3 GC) as an electrocatalyst, and we observedthat the electrochemical oxidation of FA is almost the same asthe electrochemical response of the bare GC electrode (Fig. 3A;inset). Unlike bare GC and b-Fe2O3 modied GC electrodes, theFA oxidation peak is highly stable at the a-Fe2O3 nanobermodied GC electrode. This indicated that a-Fe2O3 nanoberseffectively prevent the fouling caused by the oxidized productsof FA. The observed oxidation peak for FA in Fig. 3A is due to thetwo electron oxidation of FA to dehydrofolic acid,41 as shown inScheme 1. The oxidation process can be deduced through anelectrocatalytic mechanism involving the Fe(III)/Fe(II) ioncenters, and the catalytic mechanism of the a-Fe2O3 to folic acidoxidation can be explained by the following scheme; the vol-tammetric response of FA at the a-Fe2O3 electrode is due to twosteps, viz., an electrochemical process followed by a chemicalreaction. In the rst step, Fe(II) was electrochemically oxidizedto Fe(III) (eqn (1)) and in the second step FA was chemicallyoxidized to dehydrofolic acid by Fe(III) (eqn (2)).

2Fe(II) / 2Fe(III) + 2e� (1)

2Fe(III) + folic acid / 2Fe(II) + dehydrofolic acid + H2O (2)

The a-Fe2O3 nanober does not show any oxidation peak inthe absence of FA (curve e). Further, in order to understand the

Scheme 1 Electrochemical oxidation of FA at the a-Fe2O3 nanofiber modifiedGC electrode.

1782 | Analyst, 2013, 138, 1779–1786

fast electron transfer reaction of FA at the a-Fe2O3 nanobermodied GC electrode quantitatively, we have calculated thestandard heterogeneous rate constant (ks) for FA at a-Fe2O3

nanobers and bare GC electrodes using the Velasco equation42

as given below:

ks ¼ 1.11Do1/2 (Ep � Ep/2)

�1/2n1/2

where, ks is the standard heterogeneous rate constant; Do is theapparent diffusion coefficient; Ep is the oxidation peak poten-tial; Ep/2 is the half-wave oxidation peak potential and n is thescan rate. In order to determine ks, it is necessary to nd thediffusion coefficient for FA. The apparent diffusion coefficient(Do) value was determined using a single potential chro-noamperometry technique based on the Cottrell slope obtainedby plotting current versus 1/Otime. Chronoamperometrymeasurements were carried out for FA both at bare and a-Fe2O3

nanober modied GC electrodes aer 20 potential cycles. TheDo of 1.98 � 10�6 cm2 s�1 was obtained for FA. The estimated ksvalues for the oxidation of FA at bare and a-Fe2O3 nanobermodied GC electrodes were found to be 1.43 � 10�5 cm s�1

and 2.91 � 10�4 cm s�1, respectively. The obtained higher ksvalue for FA at the a-Fe2O3 nanober modied GC electrodeindicated that the oxidation of FA was faster at the a-Fe2O3

nanober modied GC electrode than at the bare GC electrode.Further, we have investigated whether the oxidation of FA at

the a-Fe2O3 nanober modied GC electrode is due to diffusioncontrol or adsorbed species by varying the scan rates. Theoxidation current of FA was increased while increasing the scanrates (Fig. 3B). A good linearity between the anodic peak currentand the square root of the scan rate was obtainedwithin the rangefrom 100 to 1000 mV s�1 with a correlation coefficient of 0.995for FA, as shown in the inset of Fig. 3B. This indicated that theelectrode reaction process was controlled by the diffusion of FA.

Further, we have studied the optimization of pH for thepresent FA sensor. ESI, Fig. S1A,† shows the DPVs obtained for100 mM FA at the a-Fe2O3 nanober modied GC electrode frompH 5.2–10.2 PB solution. It can be clearly visualized that as thepH value increases, the Epa of FA shis towards negativepotential, which conrms that during electrochemical oxida-tion of FA not only electrons but also protons are involved. Theplot of Epa vs. pH shows good linearity in the pH range of 5.2–10.2. The linear regression equation of Epa/V (�0.032 V) vs. pHwas obtained with a correlation coefficient r ¼ 0.990, indicatingthat the number of protons and electrons involved is equal.Fig. S1B† also reveals that the Ipa increases with an increase inpH up to 7.2, and a further increase of pH results in the decreaseof the anodic peak current. Since the present modied electrodeshows a higher current for FA at pH 7.2 and it is also close to thephysiological pH value, we have chosen pH 7.2 for the deter-mination of FA in this work.

3.3 Selective determination of FA in the presence of AA

Further, we have investigated the determination of FA in thepresence of very high concentrations of AA. It is well known thatAA is an important interferent compound which coexists withFA in our body uids, and further its concentration is always

This journal is ª The Royal Society of Chemistry 2013

Fig. 4 DPVs obtained for the increment of 10 mM FA to 2500 mM AA in a 0.2 MPB solution at the a-Fe2O3 nanofiber modified GC electrode. Pulse width¼ 0.06 s,amplitude ¼ 0.05 V, sample period ¼ 0.02 s and pulse period ¼ 0.2 s.

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much higher than that of FA.43 For example, the concentrationsof AA and FA in human blood serum are 53.8 � 36.6 mmol L�1,and 34.4 � 10.4 nmol L�1, respectively.43–45 Therefore, from aclinical point of view, the determination of FA in the presence of

Fig. 5 (A) Amperometric i–t curve for the determination of FA at the a-Fe2O3 nanconcentration of 60 nM of FA. Eapp¼ 0.90 V. (B) Calibration plot obtained for conc. of(g) 15 000, (h) 20 000, (i) 30 000, (j) 40 000 and (k) 60 000 mM addition of FA atobtained for the addition of 60 nM FA (a–c) and a mixture of 60 nM each of FA andinterval of 50 s.

This journal is ª The Royal Society of Chemistry 2013

higher concentrations of AA is very important. Fig. 4 shows theDPVs obtained for the increment of 10 mM FA in the presence of2500 mM AA. The concentration of FA was varied from 10 to50 mM (curves b–f). A very clear signal was observed for 10 mMFAin the presence of 2500 mMAA in Fig. 4 (curve b), which revealedthat detection of a very low concentration of FA is possible evenin the presence of 250-fold AA. On increment of 10 mM FA to aPB solution containing 2500 mM AA, the oxidation current of FAwas increased linearly with a correlation coefficient of 0.9995.However, the oxidation peak current of AA was almostunchanged in each addition of FA. These results demonstratedthat the a-Fe2O3 nanober modied GC electrode is moreselective towards FA even in the presence of very high concen-trations of AA.

3.4 Amperometric determination of FA along with AA

The amperometric method was used to examine the sensitivityof the a-Fe2O3 nanober modied GC electrode towards thedetection of FA individually and also along with AA. Fig. 5Ashows the amperometric i–t curve for FA at the a-Fe2O3 nano-ber modied GC electrode in a homogeneously stirred 0.20 MPB solution by applying a potential of 0.90 V. The modiedelectrode shows the initial current response due to 600 nM FA.The current response increases and a steady state current is

ofiber modified GC electrode in a 0.2 M PB solution. Each addition increases theFA vs. amperometric current. (C) (a) 60, (b) 300, (c) 900, (d) 1500, (e) 4500, (f) 9000,the a-Fe2O3 nanofiber modified electrode. (D) Amperometric i–t curve responseAA (d–f) using the a-Fe2O3 nanofiber electrode in a 0.2 M PB solution at a regular

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Table 1 Comparison of different chemically modified electrodes for the deter-mination of FA with the a-Fe2O3 nanostructure modified electrode

Modied electrodes Detection limit Ref.

Single-walled carbonnanotube-ionic liquidpaste electrode

1 � 10�9 M 46

Single-walled carbon nanotubelm modied glassycarbon electrode

1 � 10�9 M 47

Lead lm modied glassycarbon electrode

7 � 10�10 M 49

Poly(5-amino-2-mercapto-1,3,4-thiadiazole) lm modiedglassy carbon electrode

2.3 � 10�10 M 50

3-Amino-5-mercapto-1,2,4-triazolepolymerized lm modiedglassy carbon electrode

2.5 � 10�7 M 51

a-Fe2O3 nanostructure modiedglassy carbon electrode

1.12 � 10�10 M This work

Fig. 6 Amperometric i–t curve for 60 nM addition of FA at the a-Fe2O3 nanofibermodified GC electrode (a–c, g–i), and the addition of 60 mM of Na+, Ca2+, SO4

2�

(d–f), glucose, urea and oxalate (j–l), in a homogeneously stirred 0.2 M PBsolution.

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attained within 3 s for further addition of 60 nM FA in each stepwith a sample interval of 50 s. The dependence of the responsecurrent with respect to the concentration of FA was linear from60 nM to 600 nM at the a-Fe2O3 nanober modied GC elec-trode with a correlation coefficient of 0.9991 (Fig. 5B). Thecurrent response for 60 nM FA was found to be 39.2 nA. Further,the amperometric current response was increased linearly withincreasing FA concentration in the range of 60–60 000 nM(Fig. 5C) with a correlation coefficient of 0.9901, and the a-Fe2O3

nanober modied electrode displayed an excellent sensitivityfor FA detection with an experimental detection limit of 60 nM(1.12 � 10�10 M (S/N ¼ 3)). The linear range and the lowestdetection limit for FA at a-Fe2O3 nanobers were compared withthe recently reported chemically modied electrodes.46–51 Thus,the present modied electrode shows the lowest detection limitfor FA (60 nM (1.12 � 10�10 M (S/N ¼ 3)) when compared to thereported FA detection limits (see Table 1).46–51 As mentionedabove, the normal level of FA in blood serum is 34.4� 10.4 nmolL�1. Therefore, the a-Fe2O3 nanober modied GC electrode ismore suitable for the determination of FA in real (blood serum)samples even in the presence of 53.8 � 36.6 mmol L�1 AA. Theamperometric method was also performed to determine theconcentration of FA along with AA. The amperometric currentresponse for the alternative addition of AA and FA in themixture is shown in Fig. 5D. The a-Fe2O3 nanobermodied GCelectrode showed the initial current response due to the addi-tion of 60 nM AA (Fig. 5D; curves a–c) into a PB solution with asample interval of 50 s, and the current response was increased.Further, the addition of a mixture of 60 nM AA and 60 nM FA toa stirred solution of 0.2 M PB showed a two-fold enhancedamperometric oxidation current at the same applied potential(Fig. 5D; curves d–f). The two-fold amperometric oxidationcurrent obtained was due to the oxidation of both AA and FA.

3.5 Anti-interference ability of the a-Fe2O3 nanobers

The anti-interference ability of the a-Fe2O3 nanobers wastested towards the detection of FA from various common ions

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such as Na+, Ca2+, and SO42�, and some physiological interfer-

ents such as glucose, urea and oxalate using the amperometricmethod (Fig. 6). Furthermore, no change in the amperometriccurrent response was observed for 60 nM FA in the presence of60 mM of MgSO4, CaCl2, NaCl, K2CO3, NaF, Cl

�, F�, and NH4Cl,indicating that the present modied electrode is highly selectivetowards the determination of FA even in the presence of a 1000-fold excess of these interferents.

3.6 The stability and reproducibility of the a-Fe2O3

nanober modied electrode

In order to investigate the stability of the a-Fe2O3 nanobermodied GC electrode, the DPVs for 0.20 mM FA in a 0.20 M PBsolution were recorded for every 5 min interval. It was foundthat the oxidation peak current remained the same with arelative standard deviation of 2.1% for 20 repetitive measure-ments, indicating that the electrode has a good reproducibilityand does not undergo surface fouling. Aer voltammetricmeasurements, the electrode was kept in a pH¼ 7.2 PB solutionat room temperature. The current response decreased about1.24% in one week and 5.54% in about two weeks. To ascertainthe reproducibility of the results, three different GC electrodeswere modied with the a-Fe2O3 nanobers and their responsetowards the oxidation of 0.50 mM AA and FA was tested by 20repeated measurements. The separation between the voltam-metric peaks of AA–FA was the same at all the four electrodes.The peak current obtained in the 20 repeated measurements ofthree independent electrodes showed a relative standard devi-ation of 1.48%, conrming that the results are reproducible.The above results showed that the present modied electrodewas very much stable and reproducible towards these analytes.It is worthy to compare the determination of FA at the a-Fe2O3

nanober modied GC electrode with other chemically modi-ed electrodes. In the reported papers, the procedures adoptedfor the modication of electrode surfaces are very tedious, moretime consuming and further reproducible results cannot beobtained.47,48,52–54 In the case of a carbon paste electrode, rstthe carbon paste was mixed with the palmitic and stearic acids

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Fig. 7 DPVs obtained for blood serum (green line) and after the addition of10 mM commercial FA (blue line) to blood serum at the a-Fe2O3 nanofibermodified GC electrode in a 0.2 M PB solution. Pulse width ¼ 0.06 s, amplitude ¼0.05 V, sample period ¼ 0.02 s and pulse period ¼ 0.2 s.

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in the presence of carbon tetrachloride and then dried over-night at room temperature.52 Similarly, for the fabrication of amulti-walled carbon nanotube coated Au electrode, the multi-walled carbon nanotubes (MWNTs) were reuxed in the mixtureof concentrated H2SO4 and HNO3 for 4–5 h, then washed withwater and dried in vacuum at room temperature.53 Whencompared to the reported procedure for the electrode modi-cations, the procedure for the deposition of a-Fe2O3 nanoberson the GC electrode in the present study is very easy, less timeconsuming (<12 min), highly stable and reproducible.

3.7 Determination of FA in human blood serum samples

The practical application of the a-Fe2O3 nanober modied GCelectrode was tested by measuring the concentration of FA inhuman blood serum samples. The human blood serum sampleswere collected from a local hospital (Muthu clinic and X-rays,Dindigul district, India). The standard addition technique wasused for the determination of FA in serum samples. The DPV ofblood serum in a PB solution (pH ¼ 7.2) shows two oxidationpeaks at 0.35 and 0.81 V as shown in Fig. 7, green line, and thesepeaks may be due to the oxidation of AA and FA, respectively. Toconrm the observed oxidation peak at 0.81 V for FA in Fig. 7,green line, we have added a known concentration of FA into thesame blood serum solution, the oxidation current at 0.81 V wasfurther enhanced (Fig. 7; blue line) and recovery results aregiven in Table 2. The enhanced oxidation peak current at 0.81 Vindicated that the peak corresponds to the oxidation of FA. Theproposed method shows a better recovery of spiked FA in serum

Table 2 Determination of FA in human blood serum samples

Humanbloodserum

Original(mM)

Added(mM)

Found(mM)

Recovery(%)

Sample 1 50.10 10 59.80 99.5Sample 2 25.40 10 35.16 99.3

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samples, indicating that the adopted method could be effi-ciently used for the determination of FA in real samples in thepresence of possible interferents.

4 Conclusions

We have demonstrated the synthesis of a-Fe2O3 nanobers by asimple electrospinning method and their application in vol-tammetric determination of FA in the presence AA (pH 7.2). Thea-Fe2O3 nanober modied electrode not only separates thevoltammetric signals of AA and FA with a potential difference of420 mV between AA and FA, but also shows a higher oxidationcurrent than the bulk-Fe2O3 and unmodied electrodes. Theamperometric current response is linearly dependent on FAconcentration in the range of 60–60 000 nM, and the a-Fe2O3

nanober modied GC electrode displayed an excellent sensi-tivity for FA detection with an experimental detection limit of60 nM (1.12 � 10�10 M (S/N ¼ 3)). The practical application ofthe present modied electrode was successfully demonstratedby determining the concentration of FA in human blood serumsamples. The excellent analytical performance and low costnanomaterials are not only scientically signicant for thedevelopment of effective biosensors, but also could produce realbenets such as energy and cost savings in comparison withother noble metals or enzymes for a wide range of potentialapplications in medicine, catalysis, and biosensing.

Acknowledgements

The authors thank the National University of Singapore andNanyang Technological University for providing excellentresearch facilities to carry out this work. Palanisamy Kannanand Marcin Opallo thank NanOtechnology, Biomaterials andaLternative Energy Source for the ERA Integration [FP7-REGPOT-CT-2011-285949-NOBLESSE] Project from the Euro-pean Union.

References

1 J. Kong, N. R. Franklin, C. Zhou, M. G. Chapline, S. Peng,K. Cho and H. Dai, Science, 2000, 287, 622–625.

2 P. Poizot, S. Laruelle, S. Grugeon, L. Dupont andJ. M. Tarascon, Nature, 2000, 407, 496–499.

3 D. Yu and V. W.-W. Yam, J. Am. Chem. Soc., 2004, 126, 13200–13201.

4 G. Neri, A. Bonavita, S. Galvagno, P. Siciliano and S. Capone,Sens. Actuators, B, 2002, 82, 40–47.

5 Y. Cui, Q. Wei, H. Park and C. M. Lieber, Science, 2001, 293,1289–1292.

6 K. Ramanathan, M. A. Bangar, M. Yun,W. Chen, N. V. Myungand A. Mulchandani, J. Am. Chem. Soc., 2004, 127, 496–497.

7 R. J. Chen, S. Bangsaruntip, K. A. Drouvalakis, N. Wong ShiKam, M. Shim, Y. Li, W. Kim, P. J. Utz and H. Dai, Proc.Natl. Acad. Sci. U. S. A., 2003, 100, 4984–4989.

8 Y. Ding, P. X. Gao and Z. L. Wang, J. Am. Chem. Soc., 2004,126, 2066–2072.

Analyst, 2013, 138, 1779–1786 | 1785

Analyst Paper

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nloa

ded

by U

nive

rsity

of

Tex

as L

ibra

ries

on

22 F

ebru

ary

2013

Publ

ishe

d on

31

Janu

ary

2013

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/C

3AN

0007

0BView Article Online

9 J. Liu, X. Wang, Q. Peng and Y. Li, Adv. Mater., 2005, 17, 764–767.

10 X. Wang and Y. Li, J. Am. Chem. Soc., 2002, 124, 2880–2881.11 Z. W. Pan, Z. R. Dai and Z. L. Wang, Science, 2001, 291, 1947–

1949.12 C. Feldmann, Adv. Mater., 2001, 13, 1301–1303.13 W. Weiss, D. Zscherpel and R. Schlogl, Catal. Lett., 1998, 52,

215–220.14 M. Fukazawa, H. Matuzaki and K. Hara, Sens. Actuators, B,

1993, 14, 521–522.15 F. Bondioli, A. M. Ferrari, C. Leonelli and T. Manfredini,

Mater. Res. Bull., 1998, 33, 723–729.16 S. C. Tsang, V. Caps, I. Paraskevas, D. Chadwick and

D. Thompsett, Angew. Chem., Int. Ed., 2004, 43, 5645–5649.17 L. Gao, J. Zhuang, L. Nie, J. Zhang, Y. Zhang, N. Gu, T. Wang,

J. Feng, D. Yang, S. Perrett and X. Yan, Nat. Nanotechnol.,2007, 2, 577–583.

18 J. Wang, Chem. Rev., 2007, 108, 814–825.19 N. Ding, N. Yan, C. Ren and X. Chen, Anal. Chem., 2010, 82,

5897–5899.20 H.-L. Zhang, X.-Z. Zou, G.-S. Lai, D.-Y. Han and F. Wang,

Electroanalysis, 2007, 19, 1869–1874.21 G. Zhao, J.-J. Xu and H.-Y. Chen, Electrochem. Commun.,

2006, 8, 148–154.22 S.-F. Wang and Y.-M. Tan, Anal. Bioanal. Chem., 2007, 387,

703–708.23 X. Cao, N. Wang, X. Lu and L. Guo, J. Electrochem. Soc., 2010,

157, K76–K79.24 X. Cao and N. Wang, Analyst, 2011, 136, 4241–4246.25 D. Sun, H. Wang and K. Wu,Microchim. Acta, 2006, 152, 255–

260.26 T. Gunduz, E. Kiliç, E. Canel and F. Koseoglu, Anal. Chim.

Acta, 1993, 282, 489–495.27 E. Gujska and A. Kuncewicz, Eur. Food Res. Technol., 2005,

221, 208–213.28 M. O'Neil, S. Budavari, A. Smith, P. Heckelman and

J. Obenchain, Merck Index, Merck, New York, 1996, p. 715.29 P.-T. Lin, B.-J. Lee, H.-H. Chang, C.-H. Cheng, A.-J. Tsai and

Y.-C. Huang, Nutr. Res., 2006, 26, 460–466.30 M. P. McRae, Journal of Chiropractic Medicine, 2009, 8, 15–24.31 M. T. Abou-Saleh and A. Coppen, J. Psychosom. Res., 2006, 61,

285–287.32 A. H. Liem, A. J. van Boven, N. J. G. M. Veeger, A. J. Withagen,

R. M. Robles de Medina, J. G. P. Tijssen and D. J. vanVeldhuisen, Int. J. Cardiol., 2004, 93, 175–179.

1786 | Analyst, 2013, 138, 1779–1786

33 K. K. Y. Sie, J. Chen, K.-J. Sohn, R. Croxford, L. U. Thompsonand Y.-I. Kim, Cancer Lett., 2009, 280, 72–77.

34 A. T. Vasilaki, D. C. McMillan, J. Kinsella, A. Duncan,D. S. J. O'Reilly and D. Talwar, Clin. Chim. Acta, 2010, 411,1750–1755.

35 P. Torres, P. Galleguillos, E. Lissi and C. Lopez-Alarcon,Bioorg. Med. Chem., 2008, 16, 9171–9175.

36 S. Y. Ly, H. S. Yoo, J. Y. Ahn and k. h. Nam, Food Chem., 2011,127, 270–274.

37 J. Rodrıguez Flores, G. C. Pe~nalvo, A. E. Mansilla andM. J. R. Gomez, J. Chromatogr., B: Anal. Technol. Biomed.Life Sci., 2005, 819, 141–147.

38 M. J. Esteve, R. Farre, A. Frigola and J. M. Garcia-Cantabella,J. Chromatogr., B: Biomed. Sci. Appl., 1997, 688, 345–349.

39 A. Mu~noz de la Pe~na, I. D. Meras, A. Jimenez Giron andH. C. Goicoechea, Talanta, 2007, 72, 1261–1268.

40 J. Sundaramurthy, P. S. Kumar, M. Kalaivani, V. Thavasi,S. G. Mhaisalkar and S. Ramakrishna, RSC Adv., 2012, 2,8201–8208.

41 D. Dryhurst, Electrochemistry of Biological Molecules,Academic Press, New York, 1977.

42 J. G. Velasco, Electroanalysis, 1997, 9, 880–882.43 C. M. Tallaksen, T. Bøhmer and H. Bell, Am. J. Clin. Nutr.,

1992, 56, 559–564.44 A. B. Alper, W. Chen, L. Yau, S. R. Srinivasan, G. S. Berenson

and L. L. Hamm, Hypertension, 2005, 45, 34–38.45 K. S. Woo, P. Chook, Y. I. Lolin, J. E. Sanderson, C. Metreweli

and D. S. Celermajer, J. Am. Coll. Cardiol., 1999, 34, 2002–2006.

46 F. Xiao, C. Ruan, L. Liu, R. Yan, F. Zhao and B. Zeng, Sens.Actuators, B, 2008, 134, 895–901.

47 C. Wang, C. Li, L. Ting, X. Xu and C. Wang, Microchim. Acta,2006, 152, 233–238.

48 H.-S. Wang, T.-H. Li, W.-L. Jia and H.-Y. Xu, Biosens.Bioelectron., 2006, 22, 664–669.

49 M. Korolczuk and K. Tyszczuk, Electroanalysis, 2007, 19,1959–1962.

50 P. Kalimuthu and S. A. John, Biosens. Bioelectron., 2009, 24,3575–3580.

51 S. B. Revin and S. A. John, Electrochim. Acta, 2012, 75, 35–41.52 N. A. El-Maali, Biosens. Bioelectron., 1992, 27, 465–473.53 S. Wei, F. Zhao, Z. Xu and B. Zeng, Microchim. Acta, 2006,

152, 285–290.54 Q. Wan and N. Yang, J. Electroanal. Chem., 2002, 527, 131–

136.

This journal is ª The Royal Society of Chemistry 2013