Tec

SGa

b

a

ARRAA

KGPCE

1

tttppsDicstah

h0

Electrochimica Acta 139 (2014) 386–393

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

he study of adenine and guanine electrochemical oxidation usinglectrodes modified with graphene-platinum nanoparticlesomposites

tela Pruneanua,∗, Alexandru R. Birisa, Florina Pogaceana, Maria Coros a,anesh K. Kannarpadyb, Fumiya Watanabeb, Alexandru S. Birisb

National Institute for Research and Development of Isotopic and Molecular Technologies, Donath Street, No. 65-103, RO-400293 Cluj-Napoca, RomaniaCenter for Integrative Nanotechnology Sciences, University of Arkansas at Little Rock, 2801 S. University Ave., Little Rock, AR 72204, United States

r t i c l e i n f o

rticle history:eceived 14 February 2014eceived in revised form 14 May 2014ccepted 30 June 2014vailable online 17 July 2014

eywords:raphenelatinum nanoparticlesomposites

a b s t r a c t

Composite materials based on graphene and platinum nanoparticles (Gr-Pt-2 and Gr-Pt-3) were synthe-sized over two catalysts (Ptx/MgO, where x = 2 or 3 wt.%) using radio-frequency catalytic chemical vapordeposition (RF-CCVD), with methane as carbon source. After morphological (TEM/HRTEM) and struc-tural characterization (XRD, XPS, FTIR and UV-Vis) they were used to modify two gold electrodes andsubsequently employed for the investigation of adenine and guanine electrochemical oxidation. For theAu/Gr-Pt-2 electrode, the oxidation peak potential of adenine was observed at +1.19 V vs. Ag/AgCl, while,for Au/Gr-Pt-3 electrode, this was negatively shifted to +1.09 V vs. Ag/AgCl. In addition, the oxidationcurrent densities were approximately 2.7 × 10−4 and 6.9 × 10−4 A·cm−2 (for Au/Gr-Pt-2 and Au/Gr-Pt-3,respectively) demonstrating that the Gr-Pt-3 composite had a better electro-catalytic activity towards

lectrochemistry the oxidation of adenine. A similar behavior was observed for guanine oxidation. The excellent electro-catalytic properties of the Gr-Pt-3 sample were correlated with the fact that the composite material hada higher amount of platinum nanoparticles which were not fully covered by graphene layers (about 50%).In addition, it has a larger surface area (335 m2·g−1) compared with that of the Gr-Pt-2 sample (271m2·g−1), which also greatly improved the electron kinetics.

© 2014 Elsevier Ltd. All rights reserved.

. Introduction

Adenine and guanine play a fundamental role in all living sys-ems, due to their participation in processes such as cellular energyransduction or cell signaling [1]. This has led to the necessityo develop easy and efficient methods for their analysis, in com-arison with the classical methods: chemiluminescence [2], higherformance liquid chromatography [3], or isotope dilution masspectrometry [4]. The accurate determination of purine bases inNA is extremely important, since their abnormal change may

ndicate the initiation of some diseases (e.g., cancer). The electro-hemical methods offer many advantages over the classical ones,

uch as low cost, fast response, high sensitivity and real-time detec-ion [5,6]. However, the direct electrochemical oxidation of adeninend guanine cannot be recorded with bare electrodes due to theigh over-potential and slow transfer of electrons, whichgenerally

∗ Corresponding author.E-mail address: stela.pruneanu@itim-cj.ro (S. Pruneanu).

ttp://dx.doi.org/10.1016/j.electacta.2014.06.163013-4686/© 2014 Elsevier Ltd. All rights reserved.

characterize this type of electrodes. In order to overcome thesehurdles, various types of modified electrodes have been preparedand used to investigate the electrochemical oxidation of purines,such as carbon nanotubes-modified electrodes [5] or fullerene-C60-modified glassy carbon electrodes [7]. Within the last fewyears, graphene-modified electrodes have been used extensively tostudy the oxidation of various biomolecules [8,9] due to the fact thatgraphene exhibits a huge surface area along with excellent electri-cal conductivity and electron mobility at room temperature [10,11].Zhou et al. [11] have used a glassy carbon electrode modifiedwith chemically reduced graphene oxide (CR–GO/GCE) to detecta single–nucleotide polymorphism site in short oligomers, withoutany hybridization or labeling process. Xie et al. [12] have sequen-tially modified a gold electrode with a self–assembled monolayer(SAM) of n–octadecyl mercaptan and graphene sheets to obtain agraphene/SAM modified Au electrode. The graphene sheets were

randomly distributed on top of the SAM, forming nano- or sub-microarrays. The modified electrode has a wide potential windowand excellent electro-catalytic activity for the simultaneous detec-tion of all DNA bases: adenine, guanine, thymine, and cytosine.

himica

Ms

atpassa(sa

2

2

wappiiqAsosaaG

2o

i2Sds1a

rdr(t

uS1sS2Cctaprs

S. Pruneanu et al. / Electroc

oreover, the modified electrode prevents the fouling of the goldubstrate and considerably improves the electron transfer kinetics.

In this work, we present, for the first time to our best knowledge, comparative study of the electro-catalytic properties of two elec-rodes modified with composite materials based on graphene andlatinum nanoparticles. They were investigated in the presence ofdenine and guanine by using various electrochemical approaches,uch as cyclic voltammetry and linear sweep voltammetry. Thesetudies were complemented by the results obtained with otherdvanced techniques, like TEM/HRTEM, X-Ray powder diffractionXRD), X-Ray Photoelectron Spectroscopy (XPS), UV-Vis and FTIRpectroscopy, which gave us information about the morphologicalnd structural characteristics of the two composite materials.

. Experimental

.1. Preparation of graphene-platinum nanoparticles composites

Two catalysts (Ptx/MgO, where x= 2 or 3 wt.%) were prepared byet impregnation of the MgO support with H2PtCl6 (Alfa–Aesar)

queous solutions which contained the appropriate amounts oflatinum [13]. Afterward, the catalysts were dried at room tem-erature, then calcined in the air at 600 ◦C for 4 h, and next reduced

n H2 at 500 ◦C for 3 h. Graphene-platinum nanoparticles compos-tes were synthesized by radio-frequency CCVD (RF-CCVD) using auartz reactor with double walls, as described in details in ref [13].fter preparation, the samples were purified (removal of the MgOupport) by sonication at room temperature in solution of aque-us HCl (1:1) for 30 minutes and then left overnight in similar HClolution. After this process, the samples were filtered and washedbundantly with distilled water to neutral pH and dried overnightt 120 ◦C. The composite samples were denoted as Gr-Pt-2 andr-Pt-3, respectively, corresponding to each catalyst.

.2. Equipment for morphological and structural characterizationf graphene-platinum nanoparticle composites

The morphological characteristics of the purified samples werenvestigated by Transmission Electron Microscopy (JEOL JEM-100F) equipped with EDAX Genesis X-ray Energy Dispersivepectroscopy (EDS) system for elemental analysis. Specimens wereissolved in ethanol and sonicated, before a few drops of theseuspensions were dried on holey carbon girds. After drying for—15 minutes, the grids were inserted in our TEM for observationt 80 kV.

X-Ray Diffraction (XRD) measurements were performed atoom temperature and were collected in the 5 < 2�<85◦ angularomain with a Bruker D8 Advance diffractometer, using CuK�1adiation (� = 1.5406 A). In order to increase the resolution, a Ge111) monochromator in the incident beam was used to filter outhe K�2 radiation.

The chemical status of Pt in the graphene composite was studiedsing X-ray Photoelectron Spectroscopy (XPS) (K Alpha, Thermocientific). The data were collected at a background pressure of0−9 torr, using a monochromated Al K� (h� = 1436.6 eV) X-rayource. The X-ray beam used was 36 W and 400 �m in diameter.urvey scans (0 - 1350 eV) were taken at a pass energy (CAE) of00 eV and 1 eV step size. The collected data were referenced to the1s peak to 284.5 eV based on the data obtained for adventitiousarbon grown on a glass slide. Narrow scans (25 - 40 eV width) ofhe peaks of interest (C1s, Pt4f) were taken at pass energy of 50 eV

nd 0.1 eV step size to provide higher resolution analysis of theeaks. Curve fitting was performed using Powell and Simplex algo-ithms after Shirley type backgrounds were subtracted on narrowcans using the Avantage V. 5.38 software.

Acta 139 (2014) 386–393 387

Fourier Transform Infrared Spectroscopy (FTIR) measure-ments were recorded with a JASCO 6100 instrument, within4500-1000 cm−1 spectral domain with a resolution of 4 cm−1 usingKBr pellet technique.

UV-Vis spectroscopy (JASCO V-570 spectrophotometer) wasused to investigate the optical properties of Gr-Pt-2 and Gr-Pt-3suspensions (0.05 mg·mL−1 in acetate buffer pH 5).

2.3. Electrochemical measurements

Cyclic Voltammetry (CV) and Linear Sweep Voltammetry (LCV)were performed with a Potentiostat/Galvanostat Autolab-302 N(Metrohm Autolab B.V., Utrecht, The Netherlands) connected to athree electrode cell and controlled by Nova1.8 software. A gold sub-strate (0.07 cm2) was used as working electrode while a large area(approx. 2 cm2) Pt electrode was employed as counter-electrode.An Ag/AgCl electrode was used as reference. CV and LCV were typ-ically recorded between +0.2 and +1.35 V vs Ag/AgCl at a scan rateof 50 mVs−1.

2.4. Preparation of gold electrodes modified withgraphene-platinum nanoparticles composites

Prior to any modification, two gold electrodes having the samesurface area (0.07 cm2) were electrochemically cleaned in 0.5 MH2SO4 solution by cyclic voltammetry (from -0.25 to +1.6 V vsAg/AgCl; 50 cycles with a scan rate of 50 mV·s−1). Next, they wereultrasonically cleaned in both ethanol and double-distilled waterseveral times (3 minutes). Afterward, the same volume (20 �L) ofcolloidal suspension (0.5 mg·mL−1) of Gr-Pt-2 or Gr-Pt-3 in N,N-dimethyl formamide (DMF) was deposited onto each gold substrateand dried at room temperature for at least 5 hours. The modifiedelectrodes were subsequently denoted as Au/Gr-Pt-2 and Au/Gr-Pt-3, respectively.

2.5. Reagents and Solutions

The chemicals used for the electrochemical experiments wereof analytical grade. Guanine was purchased from Alfa-Aesar (SUA-Germany) while adenine was purchased from Tokyo ChemicalIndustry Co, Ltd (Tokyo, Japan). A 0.2 M acetate buffer pH 5 wasprepared from 0.2 M acid acetic and 0.2 M sodium acetate by mix-ing the appropriate volumes. Stock solutions (10−4 M guanine and10−3 M adenine) were prepared in this buffer and then diluted tolower concentrations, down to 10−6 M. N,N-dimethyl formamidewas purchased from Fluka-Germany and used for the dispersion ofthe Gr-Pt-2 and Gr-Pt-3 composites (0.5 mg·mL−1).

3. Results and discussion

3.1. Morphological and structural characterization ofgraphene-platinum nanoparticle composites

Fig. 1(a,b) presents typical TEM images of the two compositematerials (Gr-Pt-2 and Gr-Pt-3, respectively) which clearly revealthe graphene morphology. The edges of graphene layers are seen asdark lines, while the black spots represent the platinum nanoparti-cles, as indicated by X-ray Energy Dispersive Spectroscopy (XEDS)analysis (data not shown). In both cases, one can see the pres-ence of rectangular graphene flakes with variable dimensions (e.g.,42 × 44 nm; 74 × 79 nm) and a variable number of layers. The

HRTEM images in Fig. 1(c,d) present graphene flakes with 2, 5, or 6layers, indicated by the blue arrows. Moreover, Fig. 1(d) shows twometallic nanoparticles (8 and 12 nm in size) surrounded by severalgraphene layers.

388 S. Pruneanu et al. / Electrochimica Acta 139 (2014) 386–393

F r-Pt-2l

tttaoidc(t

ig. 1. TEM images of Gr-Pt-2 (a) and Gr-Pt-3 (b) composites; HRTEM images of Gayers; scale bar 2 nm (c,d).

In the case of Gr-Pt-2 sample, the nanoparticles size was foundo be within the 1-14 nm range, but most of them (about 80%) hadhe size between 2-7 nm. For the Gr-Pt-3 sample, the nanopar-icles size was also measured to be between 1 and 14 nm, withbout 78% belonging to the 2–8 nm range. These statistic data werebtained after counting over 100 nanoparticles from several TEMmages [13]. It is important to emphasize that the nanoparticle sizeistribution was relatively similar for the two samples. A major

ontribution to this could be attributed to the high heating speed350 ◦C/min), which prevents the agglomeration of Pt nanopar-icles on the MgO surface. In addition, platinum has an intense

Fig. 2. HRTEM images of Gr-Pt-2 (a) and Gr-Pt-3 (b) composites, showing the pre

(c) and Gr-Pt-3 (d) composites; the blue arrows indicate the number of graphitic

catalytic activity for reactions that involve the decomposition ofhydrocarbons [14]. Consequently, the decomposition rate ofmethane over the nanoparticles surface is very high, leading tothe formation of the first graphitic layer. This layer immobi-lizes the nanoparticles, preventing their agglomeration. By carefulexamination of a large number of TEM/HRTEM micrographs, wehave also noticed that, on a few occasions, multi-walled car-bon nanotubes (MWCNT) are also present along with graphene

flakes [13]. Fig. 2 shows such carbon nanotubes found in Gr-Pt-2 (a) and Gr-Pt-3 (b) composite samples (marked with redarrows).

sence of a few carbon nanotubes (marked with red arrows); scale bar 5 nm.

S. Pruneanu et al. / Electrochimica Acta 139 (2014) 386–393 389

0

400

800

1200

1600In

tens

ity (a

.u.)

0 2010

Gr-Pt-2

Gr-Pt-3

Gr

40302θθ (de grees )

Pt (111)

(002)

6050

Gr (100)

MgO (200)

Pt (200)Pt (220)

70 9080

Pt (311)

Fig. 3. X-ray diffraction patterns of Gr-Pt-2 (blue) and Gr-Pt-3 (red) com-pM

eifaspoatnrcsfnp

tXpptct2w

tfwc(ncPTtb

aS2d

osite, showing the characteristic peaks of both graphene and platinum.gO-characteristic peak (200) was also observed.

These results are in excellent agreement with the work of Bellt al. [15], who have reported that the size and the shape of plat-num nanoparticles supported on MgO have crucial roles in theormation of carbon nanostructures (graphene/nanotubes). In sitund ex situ TEM studies have revealed that nanoparticles with aize greater than 6 nm in diameter are generally covered by multi-le layers of graphene, the number of which depends on the timef exposure of the sample to hydrocarbon. For particles between 6nd 2 nm, carbon nanotubes are formed, while very small nanopar-icles (< 2 nm) promote the formation of graphene layers which areext transported on the MgO support. Moreover, this group alsoeported that Pt particles of similar size but with various shapesatalyzed the formation of different carbon structures under theame conditions. Consequently, there must be other contributingactors which influence the formation of either graphene or carbonanotubes. However, in our case, the synthesis process led to theredominant formation of highly crystalline graphene flakes.

XRD was used to further characterize the structure and crys-allinity of the graphene-platinum nanoparticle composites. TheRD patterns with typical diffraction lines both for graphene andlatinum are presented in Fig. 3. Here, one can see that there areeaks at around 39.7◦, 46.2◦, 67.5◦, and 80.1◦, which can be indexedo (111), (200), (220), and (311) reflections of pure Pt with face-entered-cubic (fcc) phase, respectively [16]. For Gr-Pt-3 sample,he intensity of the Pt (111) reflections is higher than that of Gr-Pt-

sample, indicating that a larger amount of platinum nanoparticlesas attached to the graphene sheets.

In addition, there are also diffraction lines which demonstratehe presence of the graphene nanostructures. The most importanteature is the intense peak centered at approximately 25.5 degreeshich can be indexed to (0 0 2) reflections in graphite. Its position

an be correlated with the distance between the graphene layersd-spacing) [17], while its intensity gives us information about theumber of layers which compose the graphene flakes [18]. In ourase, this peak is centered at 25.3 (Gr-Pt-2) and 25.6 degrees (Gr-t-3) which gives a d-spacing of 0.347 and 0.346 nm, respectively.hese values are slightly higher than the commonly reported dis-ance between graphene layers in graphite, which on average haseen found to be 0.335 nm [19].

The graphene crystallite size was estimated from the full width

t half maximum (FWHM) of the (002) diffraction peak, using thecherrer equation [20] and was determined to be 2.2 nm for Gr-Pt-

and 2.1 nm for the Gr-Pt-3 sample. By taking into account the-spacing value for each sample, we have obtained the average

Fig. 4. XPS analysis narrow scans (C1s (a) and Pt 4f (b)) collected for the Gr-Pt-3composite sample.

number of layers within the graphene flakes. For both composites,the crystallites were primarily composed of 6 layers. This is in starkcontrast with our previous work [21] in which we reported thepreparation of graphene-gold nanoparticle composites containingincreasing amounts of gold nanoparticles. In that case, the compos-ite prepared with the largest amount of gold within the catalyst (3wt% Au) had the highest degree of crystallinity and consequentlythe largest number of layers within the graphene flakes.

Next, the chemical status of platinum within the compositematerials was studied by X-ray Photoelectron Spectroscopy. Theas-collected XPS spectra of Gr-Pt-3 sample are presented in Fig. 4(a) and (b). The peak value for C–C indicates that the sp2 bonds arepredominant, while the C–OH, C–O, COOH peaks have low inten-sity and are slightly shifted towards higher binding energy values(Fig. 4a). Furthermore, the core Pt 4f spectra of Gr-Pt-3 can be fit-ted into two sets of distinct doublet peaks (Fig. 4b). The peaks weredeconvoluted into two sets of double peaks. The peaks at 71.2 eVand 74.6 eV correspond to the metallic state of Pt(0), while thoseat 72.8 eV and 76.8 eV correspond to the oxidized state of plat-inum, (Pt(II)). These peaks are consistent with platinum peaks inthe Gr-Pt-2 sample reported elsewhere [13].

Since XPS is a surface technique with a relatively limited col-

lection depth, it is very difficult to measure for such complexcomposite samples the exact ratio between the Pt in metallic andoxidized states. Some of the difficulties relate to the non-uniformityin the diameters of the Pt nanoparticles, the fact that some of them

390 S. Pruneanu et al. / Electrochimica

1000150020002500300035004000

1.5

2.0

2.5

1724

3453

1574

2919

Gr-Pt-2

Abs

orba

nce

(a.u

.)

wavenumber (cm-1 )

Gr-Pt-32850

(a)

6005004003002000.7

0.8

0.9

1.0

1.1277

277

Gr-Pt-3Abs

orba

nce

(a.u

.)

λ(nm)

Gr-Pt-2

(b)

Fig. 5. FTIR spectra of graphene-platinum nanoparticle composites: Gr-Pt-2 (blue)and Gr-Pt-3 (red) recorded within 4000-1000 cm−1 spectral domain, in KBr pellets(a); UV-Vis spectrum of Gr-Pt-2 (blue) and Gr-Pt-3 (red) suspension (0.05 mg·mL−1

il

atfpBpcewaods

aktsoiwa

ilCagn

for adenine in the case of the bare gold electrode. In the case of the

n acetate buffer pH 5) (b). (For interpretation of the references to color in this figureegend, the reader is referred to the web version of the article.)

re still incorporated in graphitic layers and that the thickness ofhe surface oxides could vary among these nanoparticles. There-ore, our approach is to compare the areas under the deconvolutedeaks (as collected within the information depths of XPS) [22,23].ased on this approach, we found that the metallic and oxidizedlatinum were in almost equal amounts (49.85:50.15%). This indi-ates that about half of the platinum corresponded to nanoparticlesntrapped between the graphene layers and was in a metallic state,hile the remaining particles were exposed to the environment

nd found to be oxidized. This is distinctly different from the statusf Pt in the Gr-Pt-2 sample, in which case the platinum was pre-ominantly in the metallic state (73%) with only 27% in the oxidizedtate [13].

In order to identify the oxygen-containing functional groupsttached to the graphene flakes, we performed FTIR analysis. It isnown that the reduced form of graphene is highly conductive, dueo the extended sp2-hybridized carbon network. In Fig. 5a, the FTIRpectra of both samples are provided and show that they exhibitnly a few vibration bands of low intensity: hydroxyl (–OH) stretch-ng band at 3450 cm−1, due to the adsorbed water molecules, along

ith those of the asymmetric/symmetric C-H stretching vibrationst 2919 and 2850 cm−1, respectively [24].

The peak at about 1724 cm−1 is attributed to the C = O stretch-ng vibrations in the carboxylic group (COOH) [24]. The extremelyow intensity of this band demonstrates that a small amount of -OOH groups is present on the graphene surface. The band located

t 1574 cm−1 corresponds to the skeletal vibrations (C = C) fromraphene sheets [25]. The FTIR spectra of the graphene-platinumanoparticle composites indicate that, after the synthesis and

Acta 139 (2014) 386–393

purification steps, the reduced form of graphene was obtained. Thisis very important for the electro-catalytic effect, since the oxygen-containing functional groups present on the graphene surface mayincrease the capacitive current, and, consequently, the electrodesensitivity is strongly diminished [26].

In order to further support that the samples are primarilycomposed of reduced graphene flakes, UV-Vis analysis was per-formed and is presented in Fig. 5b. A previous study has shownthat graphene oxide (GO) generally exhibits an absorption band ataround 231 nm (due to �-�* transitions in aromatic C = C bonds)and a shoulder at about 300 nm (due to n-�* transitions in C = Obonds [27]. After chemical reduction with hydrazine, the 300 nmshoulder disappeared, and the 231 nm band shifted to 267 nm, indi-cating the restoration of sp2 hybridization.

In our case, the Gr-Pt-2 and Gr-Pt-3 suspensions (in acetatebuffer pH5) exhibited prominent absorption bands at 277 nm,confirming that the reduced form of graphene was obtained. Noabsorption band characteristic of n-�* transitions (in C = O bonds)can be seen in these spectra. It is important to emphasize that thepresence of platinum nanoparticles attached to graphene flakeswere not evidenced by the UV-Vis analysis. One of the possiblereasons relates to the fact that many platinum nanoparticles arecovered by graphene layers (73%- for Gr-Pt-2 and 49.85% for Gr-Pt-3, as indicated by XPS analysis). The theoretical calculationsof Creighton and Eadon [28] have shown that platinum nanopar-ticles should have a plasmon peak at around 215 nm. This wasexperimentally confirmed by Henglein et al. [29] for nanoparti-cles prepared by the radiolytic reduction of PtCl24− ions, but wasnot confirmed in the case of nanoparticles prepared by reduc-tion with citrate. In fact, many other papers have reported thatthe absorbance of well-dispersed platinum nanoparticles graduallyincreased with the decrease of the wavelength, within 800-200 nmrange, without a clear absorption peak [30,31].

On the other hand, UV-Vis analysis can provide qualitative infor-mation about the amount of platinum nanoparticles attached tographene flakes. By comparing the two spectra, one can see that theabsorbance of the Gr-Pt-3 suspension is considerably lower thanthat of Gr-Pt-2, due to the fact that the Gr-Pt-3 composite has moreplatinum nanoparticles attached to graphene flakes, so the densityof this material is higher. Consequently, we can tailor the amountof metallic nanoparticles attached to graphene by simply varyingthe catalyst composition.

3.2. Electrochemical investigation of adenine and guanineoxidation using electrodes modified with graphene-platinumnanoparticles composites

After morphological and structural characterization of Gr-Pt-2and Gr-Pt-3 samples, two gold electrodes were modified by drop-casting 20 �L of each nanocomposite (0.5 mg·mL−1 in DMF) ontotheir surfaces. The electrochemical oxidation of adenine and gua-nine with bare and modified electrodes was next investigated.

A previous report has indicated that the electrochemical oxida-tion of adenine is pH dependent, and the maximum oxidation peakcurrent is obtained when the pH of the buffer solution is around5 [32]. Therefore, acetate buffer of pH 5 was selected as the sup-porting electrolyte for the following experiments. Fig. 6 shows theelectrochemical response of Au, Au/Gr-Pt-2, and Au/Gr-Pt-3 elec-trodes in an acetate buffer solution containing 3 × 10−4 M adenine.There are several marked differences between the two modifiedelectrodes and the bare gold. First of all, there is no oxidation signal

modified electrodes, there is one oxidation wave of adenine with-out the reduction, which indicates an irreversible electrochemicalprocess. For the Au/Gr-Pt-2 electrode, the oxidation peak potential

S. Pruneanu et al. / Electrochimica Acta 139 (2014) 386–393 391

1.41.21.00.80.60.40.2-3.0x10-4

0.0

3.0x10-4

6.0x10-4

9.0x10-4

1.2x10-3

1.19 V

1.09 V

AuAu/Gr-Pt-2

Au/Gr-Pt-3

E/ V vs Ag/AgCl

I /A

cm-2

3 x 10-4 M adenine

Fig. 6. Cyclic voltammograms recorded with bare gold (blue) and graphene-modified electrodes: Au/Gr-Pt-2 (black) and Au/Gr-Pt-3 (red), in acetate buffersolution pH 5 containing 3 × 10−4 M adenine; scan rate 50 mV·s−1. The currentswere normalized to the active surface area: bare Au electrode (0.07 cm2); Au/Gr-Pt-2i

oP

fic

I

wpec(

eTt

Ataowa

rrtiscrt

lsctgpctat

1.41.21.00.80.60.40.20.0

5.0x10-5

1.0x10-4

1.5x10-4

2.0x10-4

1.15 V

1.13 V

1.12 V1.11 V

Au/Gr-Pt-3 Acetate buffer pH5

10-6 M

3x10-6 M

6x10-6 M

10-5 M

3x10-5 M

6x10-5 M

10-4 M

3x10-4 M

I (A

)

E (V) vs Ag/AgCl

1.19 V(a)

0.0 2.5x10-4 5.0x10-4 7.5x10-4

0.0

2.5x10-5

5.0x10-5

7.5x10-5

y = - 4.47 x 10-7 + 0.197 CR2 = 0.997

Au/Gr-Pt-2I peak

(A)

Concentration (M)

y = - 4.927 x 10-7 + 0.476 CR2 = 0.993

Au/Gr-Pt-3(b)

Fig. 7. Linear sweep voltammograms recorded with the Au/Gr-Pt-3 electrode, inacetate buffer solution pH 5 containing various concentrations of adenine (0 -

The above electrochemical results can be interpreted in cor-

(0.113 cm2); Au/Gr-Pt-3 (0.074 cm2). (For interpretation of the references to colorn this figure legend, the reader is referred to the web version of the article.)

f adenine can be observed at +1.19 V vs. Ag/AgCl, while, for Au/Gr-t-3 electrode, this is negatively shifted to +1.09 V vs. Ag/AgCl.

We have determined the active area of the modified electrodesrom cyclic voltammetry, using Randles- Sevcik equation (1) [33],n the presence of 1 mM potassium ferrocyanide (D = 6.2 × 10−6

m2·s−1) and 0.1 M KCl (supporting electrolyte):

peak = (2.687 × 105)√

n3AC2√

Dv (1)

here Ipeak is the peak current (A), n is the number of electronsarticipating in the electrochemical process (1e−), A is the workinglectrode area (cm2), D is the diffusion coefficient (D = 6.2 × 10−6

m2/s) and C is the concentration of the potassium ferrocyanidemol/cm3).

We have obtained the followings active areas for the modifiedlectrodes: Au/Gr-Pt-2 = 0.113 cm2 and Au/Gr-Pt-3 = 0.074 cm2.he area of bare gold is 0.07 cm2. The CV’s in fig. 6 were normalizedo the active area, for each electrode.

It is interesting to emphasize that although the active area ofu/Gr-Pt-3 modified electrode is very close to that of bare gold,

he oxidation current is considerably larger. So, Gr-Pt-3 compositects as a very efficient promoter that enhances the kinetics of thexidation process. The decrease of the oxidation potential alongith the increase in the peak current density is typical behavior for

material with good electro-catalytic activity.With the increase of adenine concentration, the peak current

ecorded with the two modified electrodes also increased (see rep-esentative LCV’s for Au/Gr-Pt-3 electrode in Fig. 7a). In addition,he oxidation potential shifted slightly from 1.11 to 1.19 V. Sim-lar LCV’s were obtained for the Au/Gr-Pt-2 electrode (data nothown). Using the data obtained from various LCV’s in which theoncentration of adenine was varied from 0 to 6 × 10−4 M, the linearegression statistical analysis of peak current (Ipeak) vs. concentra-ion was obtained (see Fig. 7b).

In both cases, one can see that the calibration curves exhibitinear ranges from 0 to 10−4 M adenine; above this value, a strongaturation tendency is observed. These results can be nicelyorrelated with those of Gonc alves et al. [34], who have studiedhe oxidation of adenine on various electrodes: gold, platinum,lassy carbon, edge-plane pyrolytic graphite (EPPG), basal-planeyrolytic graphite (BPPG), and boron-doped diamond. In theirase, the best resolved peak for the oxidation of adenine was found

o occur on the EPPG electrode. The authors reported that, at a lowdenine concentration, the peak intensity was related to the oxida-ion of both adsorbed and solution phase species. At high adenine

6 × 10−4 M); scan rate 50 mV·s−1 (a); calibration plots obtained with Au/Gr-Pt-2(blue) and Au/Gr-Pt-3 (red) modified electrodes (b).

concentrations, a less electro-active polymer layer was formed onthe electrode substrate which caused a shift in the peak position.

In our case, the shift in peak potential was accompanied by adecrease in the peak current, due to the diminishing of the activesurface area. The detection limit (signal-to-noise ratio of 3) corre-sponding to the first linear range was found to be 6.3 × 10−6 M and5.8 × 10−6 M, for the Au/Gr-Pt-2 and Au/Gr-Pt-3 electrode, respec-tively.

The electrochemical behavior of guanine was next investigatedwith the two modified electrodes by Linear Sweep Voltammetry,in acetate buffer pH 5 (see representative LCV’s for the Au/Gr-Pt-2electrode in Fig. 8a; similar LCV’s were obtained for the Au/Gr-Pt-3 electrode -data not shown). As expected, in the absence ofguanine, no redox peak was observed within the potential range+0.65. . . + 1 V vs. Ag/AgCl. After guanine was added to the solution,a well- defined peak appeared at +0.83 V, which is considerablyhigher with the increased concentrations. In contrast with adenine,no shift in peak potential was observed within the 10−6 - 10−4 Mrange, which may suggest that the adsorption of guanine moleculeson graphene is considerably weaker. The calibration plots obtainedwith the Au/Gr-Pt-2 and Au/Gr-Pt-3 electrodes are represented inFig. 8b. It can be observed the presence of a small saturation ten-dency (for concentrations higher than 6 × 10−5 M), which may bedue to the adsorption of some oxidation products on the electrodesurface. The detection limit (signal-to-noise ratio of 3) for guaninewas found to be 8.7 × 10−6 M and 1.3 × 10−6 M for the Au/Gr-Pt-2and Au/Gr-Pt-3 electrodes, respectively.

relation with those obtained by Li et al. [35], who have studiedthe electrochemical oxidation of guanine in aqueous media atvarious carbon electrodes: edge plane pyrolytic graphite (EPPG),

392 S. Pruneanu et al. / Electrochimica

1.00.90.80.70.60.0

3.0x10-6

6.0x10-6

9.0x10-6

1.2x10-5

0.836 V

ace tate bu ffer pH5 10-6M 3x10-6M 6x 10-6M 10-5M 3x10-5M 6x10-5M 10-4M

I (A

)

E (V) vs Ag/AgCl

Au/Gr-Pt- 2

0.836 V

(a)

0 3x10-5 6x10-5 9x10-5 1x10-4

0

1x10-6

2x10-6

3x10-6

4x10-6

y = -1.32 x 10-8 + 0.06 CR2 = 0.98

Au/Gr-Pt-3

I peak

(A)

C (M)

y = 3.44 x 10-8 + 0.04 1 CR2 = 0.99

Au/Gr-Pt-2

(b)

Fig. 8. Linear sweep voltammograms recorded with the Au/Gr-Pt-2 electrode, ina1(

bp(psa(wnggf

bphFomeichespk

whi

[

[

[

[

cetate buffer solution pH 5 containing various concentrations of guanine (0 -0−4 M); scan rate 50 mV·s−1 (a); calibration plots obtained with the Au/Gr-Pt-2blue) and Au/Gr-Pt-3 (red) modified electrodes (b).

asal plane pyrolytic graphite (BPPG), roughened basal planeyrolytic graphite (r-BPPG), and highly ordered pyrolytic graphiteHOPG). They showed that the peak potential for the oxidationrocess decreased with the increasing number of edge planeites present at the electrode surface. Moreover, they established

correlation between the number of edge plane type defectsEPPG > r-BPPG > BPPG > HOPG) and the voltammetric peak, whichas found to increase. They concluded that there are two domi-ant factors which contribute to the electrochemical response ofuanine oxidation: (i) the density of basal plane sites available foruanine adsorption and (ii) the density of edge plane sites availableor the electrochemical oxidation.

Although it is impossible to determine the proportion ofasal/edge plane sites in graphene-platinum nanoparticles com-osites, we consider that both the Gr-Pt-2 and Gr-Pt-3 samplesave many edge planes (clearly seen in several HRTEM images–e.g.,ig. 1 c, d), which greatly promote the transfer of electrons. More-ver, the XRD analysis indicated that both samples consisted ofulti-layer (around 6) graphene flakes which, according to Güell

t al. [36], show improved heterogeneous electron transfer kinet-cs. The excellent electro-catalytic properties of the Gr-Pt-3 samplean be correlated with the fact that this composite material has aigher amount of platinum nanoparticles which are not fully cov-red by graphene layers (about 50%). In addition, it has a largerurface area (335 m2·g−1) compared with that of the Gr-Pt-2 sam-le (271 m2·g−1) [13], which also greatly improves the electroninetics.

In order to test the reproducibility of the modified electrodehich gave the best electrochemical response (Au/Gr-Pt-3) weave measured its signal in acetate buffer pH 5 solution contain-

ng 10−5 M adenine. The Relative Standard Deviation (RSD) of the

[

[

Acta 139 (2014) 386–393

oxidation peak current was determined to be 6.2% (calculated fromfive successive measurements). Moreover, the fabrication repro-ducibility was determined by modifying five gold electrodes withthe same composite material, and, in this case, the RSD was 7.1%.

4. Conclusions

In this work, we have described the synthesis by RF-CCVD ofgraphene flakes having various amounts of platinum nanoparticlesattached to the layers, using two catalytic systems: Pt(2 wt.%)/MgOand Pt(3 wt.%)/MgO. TEM/HRTEM analysis confirmed the sheet-likemorphology of the Gr-Pt-x nanostructures and evidenced the nar-row distribution of nanoparticles (over 70% were between 2 and8 nm). The crystallinity of the samples was not markedly influencedby the amount of nanoparticles attached to the graphitic layers, asproved by X-Ray powder diffraction. The Gr-Pt-2 and Gr-Pt-3 com-posites were used to modify two gold substrates and subsequentlyto detect adenine and guanine, the purine bases of DNA. The mod-ified electrodes showed a strong electro-catalytic effect, but thegreatest increase in the electrochemical signal was obtained withthe Au/Gr-Pt-3 modified electrode.

Acknowledgments

This work was supported by grants of the Romanian NationalAuthority for Scientific Research, CNCS-UEFISCDI, Project NumberPN-II-ID-PCE-2011-3-0129 and PN-II-ID-PCE-2011-3-0125. Theeditorial assistance of Dr. Marinelle Ringer is gratefully acknowl-edged.

References

[1] J. Exton, Cell signalling through guanine-nucleotide-binding regulatory pro-teins (G proteins) and phospholipases, Eur J Biochem 243 (1997) 10.

[2] M. Kai, Y. Ohkura, S. Yonekura, M. Iwasaki, Chemiluminescence determinationof guanine and its nucleosides and nucleotides using phenylglyoxal, Anal ChimActa 287 (1994) 75.

[3] B. Todd, J. Zhao, G. Fleet, HPLC measurement of guanine for the determinationof nucleic acids (RNA) in yeasts, J Microbiol Methods 22 (1995) 1.

[4] M. Hamberg, L.Y. Zhang, Quantitative determination of 8-hydroxyguanine andguanine by isotope dilution mass spectrometry, Anal Biochem 229 (1995) 336.

[5] C. Tang, U. Yogeswaran, S.M. Chen, Simultaneous determination of adenineguanine and thymine at multi-walled carbon nanotubes incorporated withpoly(new fuchsin) composite film, Anal Chim Acta 636 (2009) 19.

[6] F. Xiao, F. Zhao, J. Li, L. Liu, B. Zeng, Characterization of hydrophobic ionicliquid carbon nanotubes-gold nanoparticles composite film coated electrodeand the simultaneous voltammetric determination of guanine and adenine,Electrochim Acta 53 (2008) 7781.

[7] R. Goyal, V. Gupta, M. Oyama, N. Bachheti, Voltammetric determination ofadenosine and guanosine using fullerene-C60-modified glassy carbon elec-trode, Talanta 71 (2007) 1110.

[8] Y. Wang, Y.M. Li, L.H. Tang, J. Lu, J.H. Li, Application of graphene-modified elec-trode for selective detection of dopamine, Electrochem Commun 11 (2009)889.

[9] H. Yin, Y. Zhou, Q. Ma, S. Ai, P. Ju, L. Zhu, L. Lu, Electrochemical oxidation behaviorof guanine and adenine on graphene–Nafion composite film modified glassycarbon electrode and the simultaneous determination, Process Biochemistry45 (2010) 1707.

10] X.H. Kang, J. Wang, H. Wu, I.A. Aksay, J. Liu, Y.H. Lin, Glucose oxidase-graphene-chitosan modified electrode for direct electrochemistry and glucose sensing,Biosens Bioelectron 25 (2009) 901.

11] M. Zhou, Y.M. Zhai, S.J. Dong, Electrochemical sensing and biosensing platformbased on chemically reduced graphene oxide, Anal Chem 81 (2009) 5603.

12] X. Xie, K.K. Zhao, X.D. Xu, W.B. Zhao, S.J. Liu, Z.W. Zhu, M.X. Li, Z.J. Shi, Y.H. Shao,Study of heterogeneous electron transfer on the graphene/self–assembledmonolayer modified gold electrode by electrochemical approaches, J PhysChem C 114 (2010) 14243.

13] A.R. Biris, M.D. Lazar, S. Pruneanu, C. Neamtu, F. Watanabe, G. Kannarpady,E. Dervishi, A.S. Biris, Catalytic one-step synthesis of Pt-decorated few layergraphenes, RSC Advances 3 (2013) 26391.

14] P.N. Rylander, Catalytic hydrogenation over platinum metals, Academic Press,New York, 1967.

15] Z. Peng, F. Somodi, S. Helvig, C. Kisielowski, P. Specht, A.T. Bell, High-resolutionin situ and ex situ TEM studies on graphene formation and growth on Ptnanoparticles, Journal of Catalysis 286 (2012) 22.

himica

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

S. Pruneanu et al. / Electroc

16] Y. Si, E.T. Samulski, Exfoliated graphene separated by platinum nanoparticles,Chem Mater 20 (2008) 6792.

17] M.J. McAllister, J. Li, D.H. Adamson, H.C. Schniepp, A.A. Abdala, J. Liu, M.Herrera-Alonso, D.L. Milius, R. Car, R.K. Prudhomme, I.A. Aksay, Single sheetfunctionalized graphene by oxidation and thermal expansion of graphite,Chem. Mater. 19 (2007) 4396.

18] Z.H. Liu, Z.M. Wang, X. Yang, K. Ooi, Intercalation of organic ammonium ionsinto layered graphite oxide, Langmuir 18 (2002) 4926.

19] A. Dato, V. Radmilovic, Z. Lee, J. Phillips, M. Frenklach, Substrate-free gas-phasesynthesis of graphene sheets, Nano Lett 8 (2008) 2012.

20] H.P. Klug, L.E. Alexander, X-ray diffraction procedure for polycrystalline andamorphous materials, Wiley, New York, 1974.

21] A.R. Biris, S. Pruneanu, F. Pogacean, M.D. Lazar, G. Borodi, S. Ardelean, E.Dervishi, F. Watanabe, A.S. Biris, Few-layer graphene sheets with embeddedgold nanoparticles for electrochemical analysis of adenine, International Jour-nal of Nanomedicine 8 (2013) 1429.

22] C.J. Powell, A. Jablonski, Surface sensitivity of Auger-electron spectroscopy andX-ray photoelectron spectroscopy, Journal of Surface Analysis 17 (3) (2011)170.

23] C.J. Powell, A. Jablonski, N.I.S.T. The, electron effective-attenuation-lengthdatabase, Journal of Surface Analysis 9 (3) (2002) 322.

24] K.J. Huang, D.J. Niu, J.Y. Sun, C.H. Han, Z.W. Wu, Y.L. Li, X.Q. Xiong, Novelelectrochemical sensor based on functionalized graphene for simultaneousdetermination of adenine and guanine in DNA, Colloids and Surfaces B: Bioin-terfaces 82 (2011) 543.

25] Y. Wang, J. Liu, L. Liu, D.D. Sun, High-quality reduced graphene oxide-

nanocrystalline platinum hybrid materials prepared by simultaneous co-reduction of graphene oxide and chloroplatinic acid, Nanoscale Res Lett 6(2011) 241.

26] M. Coros, A.R. Biris, F. Pogacean, L.B. Tudoran, C. Neamtu, F. Watanabe, A.S.Biris, S. Pruneanu, Influence of chemical oxidation upon the electro-catalytic

[

Acta 139 (2014) 386–393 393

properties of graphene–gold nanoparticle composite, Electrochimica Acta 91(2013) 137.

27] F. Li, Y. Bao, J. Chai, Q. Zhang, D. Han, L. Niu, Synthesis and application of widelysoluble graphene sheets, Langmuir 26 (2010) 12314.

28] J.A. Creighton, D.G. Eadon, Ultraviolet-visible absorption spectra of the colloidalmetallic elements, J Chem Soc Faraday Trans 87 (1991) 3881.

29] A. Henglein, B.G. Ershov, M. Malow, Absorption spectrum and some chemicalreactions of colloidal platinum in aqueous solution, J Phys Chem 99 (1995)14129.

30] X. Qin, Z. Miao, X. Wang, Y. Fang, D. Zhang, Q. Chen, X. Shao, Synthesis of plat-inum nanoparticles stabilized in polyvinyl alcohol and their electrocatalyticproperties, Anal Bioanal Electrochem 3 (2011) 393.

31] R.W. Raut, A.S. Mohd, Y.S. Malghe, B.T. Nikam, S.B. Kashid, Rapid biosynthesisof platinum and palladium metal nanoparticles using root extract of Asparagusracemosus Linn, Adv Mat Lett 4 (2013) 650.

32] W. Sun, J. Liu, X. Ju, L. Zhang, X. Qi, N. Hui, Highly sensitive electrochemicaldetection of adenine on a graphene-modified carbon ionic liquid electrode,Ionics 19 (2013) 657.

33] P. Zanello, Inorganic Electrochemistry: Theory, Practice and Application TheRoyal Society of Chemistry -2003 (ISBN 0-85404-661-5).

34] L.M. Gonc alves, C. Batchelor-McAuley, A.A. Barros, R.G. Compton, Electro-chemical oxidation of adenine: A mixed adsorption and diffusion responseon an edge-plane pyrolytic graphite electrode, J Phys Chem C 114 (2010)14213.

35] Q. Li, C. Batchelor-McAuley, R.G. Compton, Electrochemical oxidation of gua-nine: Electrode reaction mechanism and tailoring carbon electrode surfaces to

switch between adsorptive and diffusional responses, J Phys Chem B 114 (2010)7423.

36] A.G. Güell, N. Ebejer, M.E. Snowden, J.V. Macpherson, P.R. Unwin, Structuralcorrelations in heterogeneous electron transfer at mono-layer and multilayergraphene electrodes, J Am Chem Soc 134 (2012) 7258.