Journal ofMaterials Chemistry B

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aInstitute of Physical Chemistry, Polish Aca

01-224 Warszawa, Poland. E-mail: ktpkann

Tel: +48 223 433 375bMaterials Science and Engineering Program

TX 78712, USA. E-mail: maiyalagan@gmailcInstitute of Materials Research and Enginee

† Electronic supplementary informa10.1039/c3tb20923g

Cite this: J. Mater. Chem. B, 2013, 1,4655

Received 1st July 2013Accepted 5th July 2013

DOI: 10.1039/c3tb20923g

www.rsc.org/MaterialsB

This journal is ª The Royal Society of

Nitrogen doped graphene nanosheet supportedplatinum nanoparticles as high performanceelectrochemical homocysteine biosensors†

Palanisamy Kannan,*a Thandavarayan Maiyalagan,*b Nanda Gopal Sahooc

and Marcin Opalloa

Functional carbon nanomaterials are significantly important for the development of high performance

sensitive and selective electrochemical biosensors. In this study, graphene supported platinum

nanoparticles (GN–PtNPs) and nitrogen doped graphene supported platinum nanoparticles (N-GN–

PtNPs) were synthesized by a simple chemical reduction method and explored as high performance

nanocatalyst supports, as well as doped nanocatalyst supports, toward electrochemical oxidation of

homocysteine (HCY) for the first the time. Our studies demonstrate that N-doped graphene supported

PtNPs show higher electrocatalytic activity for HCY with an experimental detection limit of 200 pM.

Moreover, N-doped graphene supported Pt was demonstrated to have excellent selectivity in the

electrochemical oxidation of HCY i.e., the detection of HCY is successful in the presence of a 20-fold

excess of ascorbic acid (AA). The practical application of N-doped graphene supported PtNP materials is

effectively shown for the determination of HCY in both human blood serum and urine samples, by

differential pulse voltammetry under optimized conditions. Our findings conclude that N-doped

graphene supported PtNPs can be developed as a high performance and versatile nano-electrocatalyst

for electrochemical biosensor applications.

1 Introduction

Graphene nanosheets (GNs), which are emerging as an amazingtwo-dimensional material, have been shown to have fascinatingapplications in catalysis, bioelectronics, and biosensing.1–6 Dueto its unique physical and chemical properties, such as largesurface area, tremendous conductivity and easy functionaliza-tion and fabrication, graphene provides an ideal support forelectrical and electronic devices, and biosensors.7–9 Moreover,developing the electronic characteristics of graphene to achieveunique properties has attracted great attention recently.10–12

Although, most of these approaches have been aimed atproducing graphene hybrids with synergy or multiple func-tionalities. Little attention has been paid to the intrinsicmodication of graphene for the purpose of enhancing thegraphene performance in bio-electrochemical systems.13

Chemical doping of carbon materials with hetero-atoms caneffectively tune their intrinsic properties, including electronic

demy of Sciences, 44/52 ul. Kasprzaka,

an@gmail.com; Fax: +48 223 433 333;

, The University of Texas at Austin, Austin,

.com

ring, 3 Research link, Singapore 117602

tion (ESI) available. See DOI:

Chemistry 2013

characteristics and surface structures, by causing local chemicalchanges to the elemental composition of the host material.14 Itis also known that chemical doping is a leading potentialstrategy to enrich free charge-carrier densities and enhance thethermal or electrical conductivities of materials.15–17 Forinstance, graphene can be simultaneously etched and itssurface doped with oxygen by an oxidation etching process.18

The theoretical investigation of metal doped graphene haspredicted the possibility of a Fermi level shi and a crossoverfrom p-type to n-type.19 Among the numerous potential dopants,nitrogen is considered to be an excellent element for thechemical doping of carbon materials, because it is of compa-rable atomic size and contains ve valence electrons available toform strong valence bonds with carbon atoms. As a result,nitrogen has been widely used in the doping of carbon mate-rials.20,21 Recent reports have demonstrated that nitrogendoping can signicantly increase the electron activity and alterthe electron–donor properties of carbon materials, and simul-taneously enhance the ability of carbon materials to bind withguest molecules or materials, which may lead to new propertiesin device applications.21–23

It has been reported that GNs have a relatively low density ofedge sites relative to their abundant basal plane sites.24,25 TheGNs tend to stack together because of the strong inter-sheetvan der Waals interactions. The stacking of GNs would reducetheir porosity, increase the diffusion resistance of reactants/

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electrolytes, and reduce the number of exposed active sites.Therefore, careful molecular design of GNs is needed to enabletheir use in high performance electrochemical biosensors andin other practical applications. Recently, nitrogen-doped gra-phene nanosheets (N-GNs) have been explored through chem-ical vapour deposition, and high temperature annealingmethods.14,26,27 Compared to GNs, N-GNs have a large surface-active group to volume ratio, excellent thermal stability andgood electrical and mechanical properties.28 Recent studieshave suggested that this kind of material has a high efficiencyin lithium ion batteries and super-capacitors, and also haseffective electrocatalytic activity for the oxygen reduction reac-tion.29–31 On the other hand, nanomaterials composed of mono-and bi-metals including Au, Ag, Pt and Pd have mainly beenused for applications in electrocatalysis.32 However, Lee et al.and Wang et al. have shown that PtNP–graphene nano-composites are excellent materials for the electrochemical bio-sensing of glucose and hydrogen peroxide.33,34 Ramaprabhu andco-workers successfully used nitrogen doped Pd nanoparticlesto decorate graphene nanomaterials for a renewable energyrelated application.35,36

In this paper, we report a strategy to synthesize nitrogendoped graphene nanosheet supported platinum nanoparticles(N-GN–PtNPs) and graphene nanosheet supported platinumnanoparticles (GN–PtNPs), through a simple treatment ofgraphene by ethylene glycol reduction, and further exploredthe above functionalized graphene as an efficient nano-material for the biosensing of homocysteine (HCY). We discussa simple approach for biosensing the surface modication ofHCY for the selective determination of HCY in the presence ofascorbic acid (AA) and other important interfering molecules.Interestingly, the oxidation potential of HCY at the N-GN–PtNPelectrode was shied so it was 210 mV less positive andshowed double the current density enhancement compared tothe GN–PtNP electrode. The electrical communication betweenthe platinum nanoparticles embedded in the nitrogen dopedGNs improved the electrocatalytic properties of the modiedelectrode towards HCY detection. The N-GN–PtNP electrodeshowed excellent sensitivity for HCY detection, with anexperimental detection limit of 200 pM. The present N-GN–PtNP electrode is very simple to fabricate and is stable,sensitive, and reproducible. We further demonstrated thedetermination of HCY in real samples, such as human bloodplasma and urine, using an N-doped graphene supported PtNPmodied electrode.

2 Experimental section2.1 Synthesis of graphene nanosheets (GNs)

Graphene oxide sheets were synthesized from expandablegraphite akes by a modied Hummers method.37 Briey,expandible graphite (2.0 g) was combined with 50 mL concen-trated sulfuric acid in a 250 mL beaker under vigorous agitationat room temperature. Aerwards, sodium nitrate (2.0 g) andpotassium permanganate (6.0 g) were slowly poured into thebeaker in sequence. The above mixture was heated at 35 �C for24 h and then 80 mL of distilled water was added to the

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solution. 5 min later, 20 mL of 30% H2O2 was dropped into thereaction system. Finally, the product was washed with HClsolution and then washed three times with water. The resultingsolid was dispersed in water by ultrasonication to makean aqueous dispersion of GNs with a concentration of about4 mg mL�1.

2.2 Preparation of the nitrogen doped graphene nanosheets(N-GNs)

Graphene nanosheets with a high nitrogen content weresynthesized through a one-pot process using urea as thechemical dopant.38 Typically, 10 mL graphene nanosheet(�50 mg) aqueous dispersion was diluted with 25 mL ofdeionized water, and then 2 g of urea was added to the graphenenanosheet dispersion under sonication for 3 h. Aer that, thesolution was sealed in a 50 mL Teon-lined autoclave andmaintained at 160 �C for 6 h. The solids (N-doped graphenenanosheets) were ltered and washed with distilled waterseveral times. Finally, the collected sample was dryed in avacuum oven at 60 �C to give nitrogen doped graphene. Theabove procedure is simpler and more versatile than preparingnitrogen doped graphene using a plasma treatment process.39

2.3 Preparation of nitrogen doped graphene nanosheetsupported PtNPs (N-GN–PtNPs)

Several reports in the literature discuss the deposition ofPtNPs.40–42 In this work, the functionalization of PtNPs on the N-GNs was carried out by chemical co-reduction of Pt precursorsalts along with GNs in ethylene glycol (EG)–water solutions.43,44

In brief, 100 mg nitrogen doped GNs was added to 100 mLaqueous solution containing 0.02 mM H2PtCl6, and then themixture was ultrasonically treated for 1.5 h to form a stablecolloid. Sequentially, 40 mL of EG was injected into the mixturewith magnetic stirring for 1 h and then the mixture was kept at120 �C for 6 h under magnetic stirring. The nal N-GN–PtNPcomposite was collected by ltration, washed with deionizedwater, and dried in a vacuum desiccator. Our syntheticapproach is simpler than the approach recently reported byXiong et al.45 Graphene nanosheet supported PtNPs (GN–PtNPs)were prepared using the same procedure for comparisonpurposes.

2.4 Characterization methods

The microstructure and morphology of the products wereinvestigated by eld emission scanning electron microscopy(FESEM, JEOL JSM 6301F) with an acceleration voltage of 5 kV,and high resolution transmission electron microscopy (TEM,JEOL JEM-2010) with an acceleration voltage of 200 kVmeasurements, respectively. X-ray diffraction (XRD) patternswere obtained on a Rigaku D/max-IIIB diffractometer usingCu-Ka (l ¼ 1.5406 A) at a step scan of 0.02q, from 5 to 80q.The accelerating voltage and the applied current were 40 kVand 20 mA, respectively. X-ray photoelectron spectroscopy(XPS) analysis was performed on a VG ESCALAB MK II with anMg-Ka (1253.6 eV) achromatic X-ray source. Electrochemicalmeasurements were performed in a conventional two

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compartment three electrode cell with a mirror polished 3 mmglassy carbon (GC) electrode as the working electrode, Pt wire asthe counter electrode and a NaCl saturated Ag/AgCl as thereference electrode. The electrochemical measurements werecarried out with a CHI Model 660C (Austin, TX, USA) electro-chemical workstation. In cyclic voltammetry, the electro-chemical oxidation of AA and HCY were carried out at a scanrate of 50 mV s�1. A pulse width of 0.05 s, an amplitude of0.05 V, a sample period of 0.02 s and a pulse period of 0.20 swere used in differential pulse voltammetry (DPV). For chro-noamperometric measurements, a sample interval of 0.1 s and apotential step of 0.60 mV were used. All the electrochemicalmeasurements were carried out under a nitrogen atmosphere atroom temperature (�27 �C).

3 Results and discussion3.1 Morphological characterization of the GN–PtNP andN-GN–PtNP nanocomposites

Being able to control the size and dispersion of the Pt nano-particles on graphene is very important for the application ofGN–PtNPs in fuel cells and biosensors.46,47 In our study, weprepared the PtNPs with uniform size and good distributionon the graphene nanosheets by controlling the chemicalreduction pathway in the aqueous solution. The surfacemorphology of the nanocomposite was examined by TEM andFE-SEM, XPS, and XRD measurements. It can be seen that theGN–PtNPs were transparent with voile-like structures corre-sponding to the planar graphene nanosheets. (Fig. 1A).Furthermore, the FE-SEM image of the GN–PtNPs (Fig. 1A)shows that the Pt nanoparticles were spherically shaped,highly dispersed and uniformly distributed on the GNs. Thelow magnication TEM image of the GN–PtNPs (Fig. 1B) alsoshows that the Pt nanoparticles were uniformly dispersed over

Fig. 1 FE-SEM images of GN–PtNPs (A), and nitrogen doped GN–PtNPs (C). Thecorresponding high resolution TEM images are shown in (B) and (D) respectively.A uniform dispersion of PtNPs on the GN support is clearly visible. The arrowmarks indicate the morphology of the GN-PtNP nanocomposite before and afternitrogen doping. The scale bars are 100 nm (A and C) and 50 nm (B and D).

This journal is ª The Royal Society of Chemistry 2013

the graphene nanosheets with good dispersion. The Pt nano-particles appeared as dots on the graphene nanosheets (brightand dark dots in the FE-SEM and TEM images, respectively).The distance between the particles was uniform, and theexistence of close-packed nanoelectrodes was clearly observedin the TEM image, and so it can be ascribed to the surfacefunctional groups of GNs. We considered 300 nanoparticlesand by measuring the size of the isolated particles, it has beenfound that the spherical Pt nanoparticles have a narrow sizedispersion of 8 � 0.5 nm. It has also been shown that thesurface functional groups, such as carboxyl, hydroxyl andcarbonyl groups, serve as anchoring sites for the metal nano-particles. Moreover, the oxygen functionalities, especially thosein the carboxylic acid groups, can provide active sites for thenucleation and growth of metal nanoparticles.48,49 On the otherhand, FE-SEM and TEM images of the N-GN–PtNPs (Fig. 1Cand D) show that the N-GN–PtNPs consist of randomly crum-pled sheets closely associated with each other and form adisordered solid, which might be attributed to the defectivestructure formed upon exfoliation and the presence of foreignnitrogen atoms (arrow marks). The GNs morphologies werewell maintained and clearly observed in the TEM image aernitrogen doping, indicating the high surface–volume ratio andthe two-dimensional structure of the GNs. It is clearly evi-denced that nanoparticles on the surface of the GNs do notundergo aggregation during the nitrogen doping process. Thecontent of Pt nanoparticles in the nitrogen doped GNs hybridmaterial was measured by ICP (SPS7700, Seiko) instrumentsand the ion loading was calculated as 0.063 mg cm�2. It hasbeen shown that the structure of N-GN–PtNP nanocompositesis a sandwich nanostructure, with the Pt nanoparticles notonly loading onto the surface but also in the interlayer, so thestatistics of the size distribution of the Pt nanoparticles couldnot be calculated. On the surface of the N-GN–PtNPs, the Ptnanoparticles had the same size distribution (8 � 0.5 nmdiameter) with a coverage of 92.5%. The prepared N-GN–PtNPsheets have a thickness of about �1.8 nm, with the lateral sizeranging from nanometers to several hundred micrometers. Apictorial representation showing the possible N locations inthe N-GN–PtNP nanocomposites is shown in Scheme 1.

Scheme 1 Schematic of nitrogen-doped GN–PtNPs depicting the nature of thebonding of nitrogen atoms in the graphene nanosheets.

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3.2 Structural characterization of the N-GN–PtNPnanocomposites

X-ray photoelectron spectroscopy (XPS) is a powerful tool toidentify the elements' states in bulk material.50 By the analysisof binding energy (BE) values, we have conrmed the nature ofnitrogen doping in the GN–PtNP nanosheets. Core-level high-resolution XPS spectra of the Pt (4f), N1s and C1s bindingenergies were obtained for the N-doped GN–PtNP nanosheets.Fig. 2a presents the XPS signature of the Pt (4f) doublet (4f7/2and 4f5/2). A pair of peaks with binding energies at 71.55 and74.20 eV for Pt 4f7/2 and 4f5/2 were observed, which correspondto the reduced Pt(0) nanoparticle ensemble. Considering theelectrostatic balance, the presence of positively charged Pt ionssuggests that there should be dynamic electron transfer fromthe Pt nanoparticles to the underlying GNs in the GN–PtNPnanohybrids, leading to net negative charges of the GNs. Suchan electron transfer from the nanoparticles to graphene hasrecently been conrmed by both theoretical calculations andexperimental observations.51 Furthermore, there is a 0.4 eVpositive shi of the XPS peak of the Pt(0) nanoparticles (71.5 eV)compared with bulk Pt (71.1 eV).52–55 This shi in the bindingenergy is typical for very small metal NPs on a variety of supportmaterials, and is generally attributed to reduced core–holescreening inmetal nanoparticles/clusters. This result highlightsthat the electronic properties of the Pt nanoparticle ensemble is

Fig. 2 Core level XPS spectra of (a) Pt (4f), (b) C1s, and (c) N1s orbitals of nitrogen

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signicantly different from the bulk material and biggerPtNPs,52 and such size-dependent alteration of electronicstructures likely leads to the unusual electrocatalytic properties.Fig. 2b depicts the core level XPS spectra of C1s, which shows amain peak at 284.4 eV corresponding to sp2 C1s. The smallpeaks observed at 285.5 eV and 287.1 eV correspond to theformation of N-sp2 C and N-sp3 C bonds, respectively and thiswould originate from the substitution of N atoms, defects or theedge of the graphene sheets.56–59 It has been reported that inpristine graphene, the N1s peak is absent, while in the N-dopedgraphene, the N1s peak was observed in three components(Fig. 2c), indicating that the N atoms were in three differentbonding characters inserted into the graphene nanosheets. Thetwo peaks observed at 398.37 and 400.76 eV correspond to the“pyridinic” and “pyrrolic” N atoms, respectively. These refer tothe N atoms which were located in a p conjugated system andcontribute to the p system with one or two p-electrons,respectively.56,60,61 Moreover, it has been reported that for thepyridinic-N, the nitrogen atom contributes one p-electron to thearomatic p-system and has a lone electron pair in the plane ofthe ring. As for the pyrrolic-N, the nitrogen atom contributestwo p-electrons to the p-system, and a hydrogen atom is boundin the plane of the ring, to support the graphene skeletonnanosheets.56,62 Furthermore, a peak at 401.58 eV correspondsto “graphitic” N, which refers to the N atoms that replaced the C

doped GN–PtNP nanocomposites.

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atoms inside of the graphene layers.61 Among the nitrogendoping phases (N1, N2 and N3), nitrogen was predominantlydoped in graphene in the form of pyrrolic-N, which prefers to bedoped at the edge of graphene in the presence of metal nano-particles.35 The nitrogen content within the sample has beencalculated from the XPS results and it was approximately 9.5at.%. In addition, from the XPS analysis it was observed that thecontent of pyridinic and graphitic nitrogen atoms is morewithin the GN–PtNP nanosheets.

3.3 X-ray diffraction analysis of the N-GN–PtNPnanocomposites

Fig. 3 shows the XRD pattern of N-GN–PtNP and GN–PtNPnanocomposites. The reduced graphene (GNs) showed a broaddiffraction peak observed at 27.2� in Fig. 3 (inset). This corre-sponds to the (002) plane of the graphitic carbon, and indicatesthe presence of reduced graphene in the as prepared nano-composites. This broadening of the diffraction peak suggeststhe lack of long-range ordered signatures of the graphene-basednanocomposites. The largely reduced (002) interlayer spacing of0.35 nm, in comparison with the 0.79 nm interlayer spacing ofgraphene oxide (GO), revealed that most of the oxygen func-tional groups that were intercalated into the interlayer space ofgraphite had been removed during the reduction process. It isknown that GO shows a sharp diffraction peak at 2q ¼ 11.2�,suggesting the complete exfoliation of graphite.63 Diminutionin the interplanar spacing of reduced GO as compared to GO isdue to removal of the intercalated water molecules and theoxide groups that allow graphene nanosheets to be tightlypacked.64 The relatively lower intensity peak that occurs at 43�

(2.09 A) corresponds to the (001) plane of reduced GO.65 Thenumber of layers (four) of the reduced GO has been obtainedusing the Debye–Scherrer equation.65–67

t ¼ 0.9l/b002 cos q002 (1)

n ¼ t/d002 (2)

Fig. 3 XRD patterns of GN–PtNPs (a) and N-GN–PtNP nanocomposites (b). Insetis the XRD pattern of the graphene nanosheets.

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where t is the thickness; b002 is the full width at half maximum(FWHM) corresponding to the (002) plane; n is the number ofgraphene layers and d002 is the interlayer spacing. Fig. 3a showsthat the diffraction peaks at 39.6�, 46.9�, 67.3�, 80.8� and 82.9�

are related to the (111), (200), (220), (311) and (222) planes of theface-centered-cubic (fcc) Pt (JCPDS 04-0601), conrming thatthe Pt precursor has been successfully reduced into Pt nano-particles during the course of the chemical reduction. The peakcorresponding to the (111) plane is more intense than theothers, indicating that the (111) plane is the dominatingorientation. The average crystallite size for the Pt nanoparticlesis calculated from broadening of the (111) diffraction peakusing a modied form of the Scherrer equation.65–67

d ¼ 0.9l/b1/2 cos q (3)

where d is the average particle size (nm), l is the wavelength ofthe X-ray used (1.54056 A), q is the angle at the maximum of thepeak (rad), and b1/2 is the width of the peak at half height inradians. The calculated average size of the Pt nanoparticles onthe graphene is 9 � 0.5 nm, which is closely matched with thatobtained from the FE-SEM and TEM images. Moreover, thebroad peak at 2q ¼ 27.5� is due to the (002) plane of thehexagonal structure of the graphene support, indicating thatthe nature of the GNs doesn't alter aer PtNP functionalization.The XRD diffractogram of N-GN–PtNPs is similar to that ofGN–PtNPs (Fig. 3b). For N-GN–PtNPs, the interlayer spacing isabout 3.41 A, which is little bigger than that of reduced GNs(3.36 A). This may be due to defects resulting from nitrogendoping. However, the N-doping treatment process cannot affectthe layers of the GNs.

3.4 Electrochemical oxidation of HCY at a N-GN–PtNPnanocomposite electrode

HCY is an important amino acid, which isn't found directly inthe diet, but is formed during methionine metabolism.68 It hasbeen documented that the HCY concentration in blood plasmais approximately 5–16 mmol L�1, and higher concentrations ofHCY give rise to hyperhomocysteinemia (#100 mmol L�1) orhomocystinuria (�500 mmol L�1).68 It has been shown thathyperhomocysteinemia is associated with folate and cobal-amine deciencies, and can lead to early pregnancy loss, mentaldisorders and tumors.69 Furthermore, a moderate increase inHCY concentration is associated with an increased risk ofcoronary artery and cerebrovascular diseases,70 includingatherosclerosis and thrombosis.71 Ascorbic acid (AA) always co-exists with HCY in our body uids, but its concentration is verymuch higher when compared to HCY concentration. Sincethese biomolecules coexist in human uids, their simultaneousdetermination is essential to secure human health from the riskof the above critical diseases. Therefore, highly selective andsensitive determination of HCY is very important from clinicaland health viewpoints.

We have examined the electrocatalytic activity of N-GN–PtNPGC, GN–PtNP GC, GN GC and unmodied GC electrodestowards the oxidation of HCY. We found that the N-GN–PtNPmodied GC electrode showed higher electrocatalytic activity

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towards HCY and AA than the other modied and unmodiedGC electrodes. Fig. 4A shows the cyclic voltammograms (CVs)obtained for 0.2 mM HCY at N-GN–PtNP GC, GN–PtNP GC, GNGC and bare GC electrodes in a 0.20 M phosphate buffer (PB)solution (pH ¼ 7.2). The bare GC electrode does not show anyresponse for HCY in the potential window of �0.40 to 1.00 V(curve a), while the GN modied GC electrode showed anoxidation wave for HCY at 0.63 V (curve b). Notably, the incor-poration of PtNPs into the GNs (GN–PtNPs) signicantlyincreased the oxidation current of HCY at the same potential(0.63 V; curve c). Interestingly, the N-GN–PtNP modied GCelectrode showed a 210 mV less positive potential shi (i.e.,oxidized 0.42 V) and �3-fold higher oxidation current for HCY(curve d) when compared to the GN–PtNP modied electrodeprepared under identical conditions (curve c). The observedoxidation potential (0.42 V) is comparably less positivecompared to the previously reported polymer, polymer-nano-particle and other chemically modied electrodes (referencesare listed in the ESI†). For a comparison, we performed oxida-tion of HCY using a N-GN modied electrode (curve f) andobserved that the oxidation potential of HCY shied �160 mVless positively; however the oxidation current density isincreased a small amount compared to GN–PtNPs. Thus the N-GN–PtNP composite nanomaterial is very important for devel-oping an electrochemical HCY biosensor. The N-GN–PtNPelectrode exhibited an enhanced electrocatalytic response toHCY mainly due to the following reasons. The incorporation ofnitrogen into carbon materials, especially in the form of pyr-idinium moieties,72 is critical for the enhancement of the elec-trocatalytic activity, and the nitrogen atoms doped in graphenewere predominantly pyridinic N and pyrrolic N in the nano-composite. In addition, some nitrogen atoms inserted into thegraphite plane, bonded to three carbon atoms and formedquaternary N, which is referred to as “graphitic nitrogen”(G-N).39 As has been reported, nitrogen doping introducesatomic charge density and asymmetry into the spin density onthe graphene network, which facilitates the charge transferfrom the carbon support to the adsorbing molecules. Under

Fig. 4 (A) CVs obtained for 0.2 mM HCYat (a) a bare GC electrode and (b) GN, (c) GN-GNs) at a scan rate of 50 mV s�1. (e) CV of a N-GN–PtNP modified GC electrode inoxidation of HCY. (B) Linear sweep voltammograms (LSVs) obtained for 0.2 mM HCY25, (b) 50, (c) 75, (d) 100, (e) 125, (f) 150, (g) 175, (h) 200, (i) 225 and (j) 250 mV s�1. Tthe anodic oxidation current of HCY.

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near-physiological conditions (pH ¼ 7.2), HCY has an aminogroup –NH3

+ and a carboxyl group –CO2�.73,74 At this point, the

electrostatic interaction between the negatively charged –CO2�

group of HCY and the positively charged pyridiniummoieties ofthe N-doped GN–PtNPs gave a higher oxidation current for HCY.It has also been implied that the incorporated nitrogen atomscan enhance the interaction between the carbon structure andmetals, thus the kinetics of HCY diffusion and biosensing effi-ciency can be improved. Moreover, PtNPs have a large specicactive surface area and can act as tiny conducting centers, whichwere distributed throughout the GN network and formed acontinuous assembly of PtNPs on the surface, resulting in adecrease in the energy barrier, facilitating electron transfer, andimproving biosensor response. It has been shown that the thiol(–SH) and amino group (–NH3

+) in the HCY molecule have ahigher affinity for PtNPs in the nanocomposite, through Pt–Sand interparticle binding and electrostatic interactions betweenamino groups. This means that more HCY biomolecules canaccess the PtNP surface by three-dimensional assembly(Scheme 2), as compared to the two-dimensional substrate. Tounderstand the fast electron transfer reaction of HCY at the N-GN–PtNP modied electrode quantitatively, we have calculatedthe standard heterogeneous rate constant (ks) for HCY at N-GN–PtNP modied electrodes and other electrodes. HCY oxidationis an irreversible process and hence we have used Velasco eqn(4) (ref. 75) to calculate the heterogeneous rate constant (ks):

ks ¼ 1.11Do1/2(Ep � Ep1/2)

�1/2y1/2 (4)

where ks is the standard heterogeneous rate constant; Do is theapparent diffusion coefficient; Ep is the oxidation peak poten-tial; Ep1/2 is the half-wave oxidation peak potential and y is thescan rate. In order to determine the ks, it is essential to nd thediffusion coefficient values for HCY. The Do value was deter-mined by a single step potential chronoamperometry methodbased on the Cottrell slope obtained76 by plotting current versus1/Otime. The chronoamperometry measurements were per-formed for HCY at GN, GN–PtNP and N-GN–PtNP modied

N–PtNP and (d) N-GN–PtNP modified GC electrodes in 0.2 M PB solution (pH ¼ 7.2the absence of 0.2 mM HCY. (f) CV of a N-GN modified GC electrode toward theat a N-GN–PtNP modified electrode in 0.2 M PB solution at different scan rates: (a)he inset calibration plot is obtained by plotting the square root of the scan rate vs.

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Scheme 2 Schematic representation of electrostatic, amino-Pt and thiol-Ptinteractions between N-GN–PtNPs and HCY molecules.

Fig. 5 CVs obtained for a mixture of 0.2 mM each of HCYand AA at (a) a bare GCelectrode and (b) GN, (c) GN–PtNP and (d) N-GN–PtNP modified GC electrodes in0.2 M PB solution (pH ¼ 7.2) at a scan rate of 50 mV s�1. Curve (e) corresponds tothe CV of a N-GN–PtNP modified GC electrode after the 20th measurement and(f) is the CV of a N-GN–PtNP modified GC electrode in the absence of HCYand AA.

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electrodes aer 20 potential cycles and Do values of 2.85 � 10�5

cm2 s�1, 4.96 � 10�5 cm2 s�1 and 8.12 � 10�5 cm2 s�1 wereobtained for GN, GN–PtNP and N-GN–PtNP modied GC elec-trodes, respectively. The estimated ks values for the oxidation ofHCY at GN, GN–PtNP and N-GN–PtNP modied GC electrodeswere found to be 1.84 � 10�3, 3.27 � 10�3 and 8.74 � 10�3 cms�1, respectively. The bare GC electrode failed to oxidize theHCY and thus the ks value was not obtained for the bare GCelectrode. The higher ks value for HCY at the N-GN–PtNPmodied electrode indicated that the oxidation of HCY wasfaster at N-GN–PtNP modied electrode than the GN–PtNP, GNand bare GC electrodes. Furthermore, we recorded linear sweepvoltammograms (LSVs) at different scan rates to show whetherthe oxidation of HCY at the N-GN–PtNP modied electrode isdue to diffusion control or adsorption. The oxidation peakcurrent of HCY increased while increasing the scan rate(Fig. 4B). A good linearity between the anodic peak current andthe square root of the scan rate, with a correlation coefficient of0.9910, was obtained within the range of 25–250 mV s�1. Thisindicated that the electrode reaction of HCY was under diffu-sion control.

3.5 Simultaneous determination of HCY and AA

It is essential to determine HCY in the presence of AA becauseAA is one of the main interferents for the determination of HCY.The CVs obtained for a mixture of 0.2 mM each of HCY and AAat a bare GC electrode and at GN, GN–PtNP and N-GN–PtNPmodied electrodes in 0.2 M PB solution (pH ¼ 7.20) are shownin Fig. 5. The bare GC electrode failed to separate the voltam-metric signals of AA and HCY, as evidenced from the mixedvoltammetric wave around 0.27 V (curve a). However, the GNmodied electrode resolves the oxidation peaks of HCY and AAat 0.65 and 0.05 V with a potential difference of 600 mV (curveb). Then, the incorporation of PtNPs into the GNs signicantlyincreased the oxidation current of both HCY and AA (curve c) atthe same potential. Interestingly, the N-GN–PtNP modiedelectrode oxidizes HCY and AA at 0.50 and�0.12 V, respectively.

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It has been observed that the N-GN–PtNP modied electrodeshowed nearly 3-fold-enhancement in the oxidation current ofHCY and AA, and signicantly reduced the oxidation over-potential to about �160 mV less than the GN–PtNP modiedelectrode. In addition, the oxidation peaks of both HCY and AAwere highly stable even aer 20 cycles, as evidenced in curve e.The N-GN–PtNP modied electrode did not show any voltam-metric response in the absence of HCY and AA (curve f).Furthermore, this N-GN–PtNP modied electrode showed a verylarge peak separation (620 mV) between AA and HCY at physi-ological pH. Usually higher concentrations of AA co-exists withHCY in our body uids, therefore it is highly essential todetermine HCY in the presence of a higher concentration of AA.The differential pulse voltammograms (DPVs) obtained for theoxidation of 10 mMHCY in the presence of 0.2 mM AA is shownin Fig. 6. A clear voltammetric signal was observed for HCY evenin the presence of a 20-fold excess of AA (curve b). The additionof each 10 mM HCY to 0.2 mM AA in PB solution increases thecurrent due to the oxidation of HCY, while the peak current dueto AA was unchanged. The observed results indicate that theN-GN–PtNP modied electrode is more sensitive towards theoxidation of HCY, even in the presence of higher concentrationsof AA (20-fold excess). The oxidation peak currents of HCYlinearly increased in each 10 mM addition of HCY with acorrelation coefficient of 0.9951. On the other hand, we testedthe same experiment for GN–PtNP modied electrode and itdidn't show an enhanced signal for HCY in the presence of AA(ESI, Fig. S1†).

3.6 Amperometric determination of HCY

The sensitivity of the N-GN–PtNP modied electrode to HCYwas analyzed using constant potential amperometry understeady state conditions. Fig. 7A depicts the amperometric i–tcurve obtained for the oxidation of HCY at the N-GN–PtNPmodied electrode in a constantly stirred 0.2 M PB solution,at an applied potential of +0.60 V. An initial steady-state

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Fig. 6 DPVs for the oxidation of HCY at a N-GN–PtNP modified GC electrode indifferent concentrations, (a) 0, (b) 10, (c) 20, (d) 30, (e) 40, (f) 50, (g) 60, and (h) 70mM, in the presence of 0.2 mM of AA in 0.2 M PB solution. Pulse width ¼ 0.05 s,amplitude ¼ 0.05 V, sample period ¼ 0.02 s and pulse period ¼ 0.2 s. Inset: cali-bration plot obtained for the concentration of HCY vs. anodic oxidation currents.

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amperometric current response was observed due to the addi-tion of 2 nMHCY. Then with the addition of a further 2 nMHCYin each step, with a sample interval of 50 s, the current responselinearly increased and a steady state current response wasobtained within 2 s, which indicates a fast electron-transferprocess at this electrode. We found that a 2.40 nA current wasobtained for the single addition of 2 nM HCY (1.35 nA nM�1) atthe N-GN–PtNP electrode. The observed stable amperometriccurrent response together with higher sensitivity to HCY of theN-GN–PtNP modied electrode indicates that this electrode canbe successfully used for the sensitive detection of 2 nM HCY.The amperometric current responses increased linearly withHCY concentration from 2 to 24 nM (Fig. 7A; curve a) and thecalibration plot obtained for amperometric current responsesvs. various concentrations of HCY is shown in the inset ofFig. 7A. A good linearity was obtained with a correlation coef-cient of 0.9970 in the concentration range that was used in

Fig. 7 (A) Amperometric i–t curves for each addition of 2 nM HCY at N-GN–PtNPregular time interval of 50 s. (B) The N-GN–PtNP electrode with 200 pM of HCY addpolarized at +0.60 V. The inset shows the corresponding calibration plot.

4662 | J. Mater. Chem. B, 2013, 1, 4655–4666

Fig. 7. For comparison, the sensitivity of the GN–PtNP modiedelectrode towards HCY was examined by amperometry and thesensitivity limit was found to be 0.6 nA nM�1 (Fig. 7 curve b).Moreover, we have observed amperometric signals with pico-molar concentrations of HCY, as shown in Fig. 7B. The N-GN–PtNP electrode was polarized at +0.60 V, and aliquots of HCYwere injected into the stirred electrolyte solution. A rapidincrease in the current was noticed aer each addition of 200pM HCY into the electrolyte solution, and a steady stateresponse was attained within 2 s. Thus the experimentaldetection limit of the present N-GN–PtNP modied electrodewas calculated to be 200 pM. The N-GN–PtNP electrode is highlysensitive, and the amperometric response is very stable andoffers a linear dependence over a wide range of HCY concen-trations (�1 mM). The observed results indicate that the N-GN–PtNPmodied electrode showed higher sensitivity than the GN–PtNP modied electrode. It is worth comparing the perfor-mance of the N-GN–PtNP modied electrode towards HCY withthose of recently reported polymer, polymer-nanoparticles andother chemically modied electrodes. We summarized thelimits of detection with relevant references in ESI, Table S1.†Our limit of detection is very low (200 pM), compared to thereports available in the literature based on other electro-chemical HCY biosensors. In our system, PtNPs were embeddedthree dimensionally onto the surface of the nitrogen dopedGNs, leading to the formation of more electroactive graphenelayers and electrostatic interactions between HCY and the threedimensionally formed N-GN–PtNP, which offers a high perfor-mance platform toward the detection of HCY.

3.7 Effect of interferents

We studied the determination of HCY in the presence ofcommon interferents such as glucose, urea, uric acid, serotoninand oxalate, by an amperometric method. The amperometric i–tcurve obtained for HCY at the N-GN–PtNPmodied electrode inthe presence of common interferents, in a constantly stirred0.2 M PB solution (pH ¼ 7.2), at a constant applied potential of+0.60 V, is shown in Fig. 8. The increased initial current

(a) and GN–PtNP (b) modified GC electrodes in 0.2 M PB solution (pH ¼ 7.2) at aed into the stirred PB solution at regular time interval of 50 s. The electrode was

This journal is ª The Royal Society of Chemistry 2013

Fig. 8 Amperometric i–t curve responses (at a constant working potential of+0.60 V vs.Ag/AgCl) obtained for 2 nMHCY (a and b) and 1 mMof glucose (c), urea(d), uric acid (e), serotonin (f), oxalate (g) and thiourea (h), additionsof 2nMHCY (i, jand k) at the N-GNs/PtNPs modified GC electrode in 0.2 M PB solution (pH ¼ 7.2).

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response was due to the addition of 2 nM HCY (a) and with thefurther addition of 2 nM HCY in each step with a sampleinterval of 100 s, the current response increases and a steadystate current response was attained within 2 s (a and b). Aertwo steps (a and b),1 mM each of glucose (c), urea (d), uric acid(e) serotonin (f), oxalate (g) and thiourea (h) were added sepa-rately with a sample interval of 50 s to the same 0.2 M PBsolution (pH ¼ 7.2), no change in amperometric currentresponse was observed. However, with the addition of 2 nMHCY to the same solution (i, j and k), the current response wasagain increased similar to steps a and b. Similarly, the selec-tivity of HCY was also studied in the presence of some otherimportant biomolecules (interferents) such as dopamine,epinephrine and L-dopa (Fig. S2 in ESI†); the N-GN–PtNPmodied electrode was highly selective toward the determina-tion of HCY in the presence of important biomolecules. Theobserved results indicate that the N-GN–PtNP modied elec-trode can be successfully used for the determination of 2 nMHCY, even in the presence of a 500-fold excess of severalcommon interferents.

Fig. 9 Plot of current vs. time for the oxidation of HCY (0.2 mM) at the N-GN–PtNP electrode in 0.2 M PB solution, demonstrating the operational stability of theelectrode.

3.8 Stability and reproducibility

The long-term storage and operational stability of the electrodeis essential for the continuous monitoring of HCY. The stabilityof the present electrode was examined by using the same N-GN–PtNP modied electrode for 20 repetitive measurements in asupporting electrolyte solution containing 0.2 mM HCY. Theelectrode used in this measurement was kept in 0.2 M PBsolution and was subjected to another 20 repetitive measure-ments aer 12 and 24 h. We observed no noticeable change inthe peak potential and peak current for the oxidation of HCY inboth sets of experiments. The coefficient of variation wascalculated separately for the two sets of experiments and we

This journal is ª The Royal Society of Chemistry 2013

found that it remained the same (0.52 and 0.63%) in both sets.This shows that the electrode is stable and does not undergopoisoning by the oxidation products, and so can be used for therepeated measurement of HCY. To further ascertain the oper-ational stability of the present electrode, a voltammetricmeasurement in a supporting electrolyte solution containing0.2 mMHCY was performed with the N-GN–PtNP electrode, andthe peak current for the oxidation of HCY was measured atregular intervals over a period of 25 h. As shown in Fig. 9, themagnitude of the peak current did not change appreciablyduring the whole set of experiments (25 h), demonstrating thatthe N-GN–PtNP electrode is very stable and retains its sensitivitythroughout the experiments. To check the long-term storagestability of the present HCY sensor, the N-GN–PtNP modiedelectrode was kept in 0.2 M PB solution (pH ¼ 7.2) at roomtemperature. No appreciable decrease in the oxidation currentresponse of HCY was observed for 4 days, and the currentdecreased by only 2.8% aer two weeks. To ascertain thereproducibility of the results, two different GC electrodes weremodied with the N-GN–PtNPs in the same way and eachelectrode response towards 0.2 mM HCY was tested by 10repeated measurements. The peak current obtained for the twoindependent electrodes again showed an RSD of 0.8%, con-rming that the results were highly reproducible.

3.9 Determination of HCY in real sample analysis

The practical application of the N-GN–PtNP modied electrodefor the determination of HCY in real samples was tested bymeasuring the concentration of HCY in human blood serumand urine samples. The human urine samples were diluted 100-fold in 0.2 M PB solution, without any other treatment thatcould reduce the matrix effect of real samples. The DPVobtained for the N-GN–PtNPmodied electrode in 0.5 mL of thehuman blood serum sample in 9.5 mL of 0.2 M PB solution isshown as line a in Fig. 10A. It shows an oxidation peak at 0.45 V,which is attributed to the oxidation of HCY. To conrm that theobserved oxidation peak was due to HCY, the sample was spiked

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Fig. 10 (A) DPVs obtained (a) for human blood serum and (b) after the addition of 20 mM commercial HCY to human blood serum, for the N-GN–PtNP modifiedelectrode in 0.2 M PB solution. (B) DPVs obtained (a) for human urine and (b) after the addition of 20 mM commercial HCY to human urine, for the N-GN–PtNP modifiedelectrode in 0.2 M PB solution from 0 to 0.70 V. Pulse width ¼ 0.05 s, amplitude ¼ 0.05 V, sample period ¼ 0.02 s and pulse period ¼ 0.2 s.

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with 20 mM commercial HCY and the resulting DPV is shown asline b in Fig. 10A. An increase in the peak current conrmedthat the oxidation peak at 0.45 V (line a) was due to the oxida-tion of HCY. The DPV experiment was performed for foursamples of human serum and the results are given in Table S2.†The recovery rates of the spiked samples were 99.8, 99.8, 99.7and 99.6% for the four samples of human blood serum (TableS2†). In addition to human blood serum samples, the applica-tion of the electrode was extended to the measurement of HCYin human urine samples. Curve a in Fig. 10B shows the DPVobtained for 10 mL of human urine sample diluted in 0.2 M PBsolution. A broad oxidation peak was observed at 0.48 V, whichis attributed to the oxidation of HCY. To conrm the observedoxidation peak was due to the oxidation of HCY, the sample wasspiked with 20 mM commercial HCY and the resulting DPV isshown as curve b in Fig. 10B. An increase in the peak currentconrmed that the oxidation peak at 0.48 V (curve b) was due tothe oxidation of HCY. The results obtained in the present studyillustrate that the N-GN–PtNP modied electrode is highlysuitable for the determination of HCY in human blood serumand urine samples.

4 Conclusions

We demonstrated the synthesis of nitrogen doped graphenesupported PtNPs (N-GN–PtNPs) as graphene based nano-composites for use as high performance electrocatalyststowards electrochemical oxidation of homocysteine (HCY). Ourinvestigation demonstrate that N-doped graphene supportedPtNPs showed higher electrocatalytic activity for HCY with anexperimental detection limit of 200 pM. In addition, the N-doped graphene supported PtNP nanocomposites were shownto have excellent selectivity for the electrochemical oxidation ofHCY, i.e., the detection of HCY is successful in the presence ofascorbic acid (AA) and other important interferents. The prac-tical application of the N-GN–PtNP nanocomposite wassuccessfully used for the determination of HCY in both human

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blood serum and urine samples by differential pulse voltam-metry under optimized conditions. Our ndings conclude thatN-GN–PtNP nanocomposites can be developed as efficient andversatile high performance electrocatalysts for electrochemicalbiosensor applications.

Acknowledgements

Palanisamy Kannan and Marcin Opallo thank NanOtechnologyBiomaterials and aLternative Energy Source for ERA Integration[FP7-REGPOT-CT-2011-285949-NOBLESSE] Project from Euro-pean Union.

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