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Electrochimica Acta 128 (2014) 128–137

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

odulation of the matrix effect of nafion on tris(bipyridine)uthenium(II) electrochemical probes by functionalisation with-nitrophenylazo graphene-gold nanocomposite

hristopher E. Sundaya, Mawethu Bilibanaa, Sinazo Qakalaa, Oluwakemi Tovidea,erileng M. Molapoa, Gertrude Fomoa, Chinwe O. Ikpoa, Tesfaye Waryoa,cineka Mbambisaa, Bulelwa Mpushea, Avril Williamsa,b, Priscilla G.L. Bakera,ibulelo Vilakazi c, Robert Tshikhudoc, Emmanuel I. Iwuohaa,∗

SensorLab, Department of Chemistry, University of Western Cape, Robert Sobukwe Road, Bellville, Cape Town 7535, South AfricaDepartment of Biological and Chemical Sciences, University of the West Indies, Cave Hill BarbadosNanotechnology Innovation Centre, Advanced Materials Division, MINTEK, Randburg, South Africa

r t i c l e i n f o

rticle history:eceived 24 July 2013eceived in revised form7 December 2013ccepted 19 December 2013vailable online 7 January 2014

a b s t r a c t

Charge transfer reactions of electroactive reagents in pure Nafion film are generally slow due to Nafion’scompact nature and the poor diffusion of ionic species within the film. Cationic reagents, such astris(bipyridine)ruthenium(II) ion ([Ru(bpy)3]2+), migrate into the electro-inactive hydrophobic regionof the ionomer causing a loss in the electrochemical contact of the cationic material with the electrode.A highly dispersive gold nanoparticle (AuNp) dotted 4-nitrophenylazo (NPA) functionalised graphene(G) nanocomposite (AuNp/G/NPA) has been prepared and incorporated into Nafion on a glassy carbon

eywords:anomaterialsrapheneraphene Oxideafion

(GC) electrode surface, in order to improve the electroactivity of the cationic reactant within the Nafionfilm. The Nafion nanocomposite-modified electrode efficiently loaded large amount of [Ru(bpy)3]2+

cationic redox probe. The sensitivity of the functionalised Nafion electrode was determined by therate at which the Ru2+ sites were regenerated within the film. In comparison to pure Nafion film, theAuNp/G/NPA/Nafion nanocomposite film exhibited 100% relative electroactivity, 30% increase in peak

tion i

ris(bipyridine) ruthenium(II) ion currents and 34.9% reduc

. Introduction

The design of an optimum interface between the transducerurface, detector material and bio-components is a key part of alllectrochemical sensor development [1,2]. Many signal amplifica-ion strategies have been applied in designing sensor platformsith optimized sensitivity, specificity and stability. These strategies

nclude applying new redox-active probes [3], coupling conductingolymers with electrochemical detection probes [4], incorporat-

ng nano-structured materials to increase loading of tags etc.mong these strategies, nano-structured materials such as metal-

ic nanoparticles, quantum dots [5], carbon nano-tubes, graphenetc., present very unique optical, magnetic, catalytic and elec-rochemical properties [6]. These unprecedented properties of

ano-structured materials have fostered their use in data storageevices, heterogeneous catalysts, chemical sensors, optoelectro-ics, optical markers, drug deliverers, fuel cells, nano-reactors

∗ Corresponding author.E-mail address: eiwuoha@uwc.ac.za (E.I. Iwuoha).

013-4686/$ – see front matter © 2014 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.electacta.2013.12.143

n charge transfer resistance.© 2014 Elsevier Ltd. All rights reserved.

and water-treatment devices among many other applications[5,7,8].

Gold nanoparticles can improve the quantity of immobilizedsignal probes due its large specific surface area, high surfaceadsorption, good stability and biocompatibility [9]. These improvedproperties are critically sensitive to their chemical structure, com-position and particle sizes [10–12]. Graphene is a monolayer ofsp2-bonded carbon atoms tightly packed into a two-dimensional(2-D) lattice. Due to its huge surface area (2600 m2/g), highchemical stability and electronic properties, it serves as scaffoldto anchor large amount of nanoparticles and also assist in pro-moting selectivity and efficiency of catalytic processes [13]. Theelectrical conductivity of graphene can be enhanced by absorbednanoparticles acting as donors or acceptors. The enhanced elec-trical conductivity and mechanical properties make graphene anexcellent material for collecting and transporting charge in photo-electrochemical solar cells, photo-catalysis and electrochemical

sensors [14,15].

Ideally, in the preparation of graphene from graphite oxide,the graphite oxide is rigorously reduced after exfoliation in orderto obtain the desirable properties of graphene. The reduction of

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C.E. Sunday et al. / Electroc

xfoliated graphite oxide leads to graphene nano-sheets withimited water solubility (< 0.5 mg/mL). Therefore solubility of pris-ine graphene is a challenge in its effective application. In other tobtain water-soluble graphene sheets in this study, graphene oxideas reduced in three stages following the method of Yongchao Si

nd Edward T. Samulski [16]. Residual oxygen functionality wasemoved and 4-nitrophenyl diazonium tetrafluoroborate was usedo introduce negatively charged 4-nitrophenylazo (NPA) units intoartially reduced graphene oxide. The presence of NPA units onraphitic sheets prevents it from aggregating in solution after thenal reduction stage of the graphene oxide because of electro-tatic repulsion between the graphene sheets, thereby yieldingsolated sheets with highly improved water solubility depicted as-nitrophenylazo functionalised graphene (G/NPA).

Nafion is an anionic polymer (a perfluorosulfonate ionomer)nd can bind molecules through coulombic and/or hydrophobicnteractions [17–19]. It can facilitate electron transfer from annzyme active centre to the electrode surface and acts as a mediatoror redox-active probes [20]. However the rate of charge transfern pure Nafion film is relatively slow due to its compact nature

hich is unfavourable for diffusion of analyte. Immobilised cationiceactants such as tris(bipyridine) ruthenium (II) ([Ru(bpy)3]2+),igrates into electro-inactive hydrophobic region of the ionomerith time, thereby losing electrochemical contact with the elec-

rode.The aim of this study is to improve the behaviour of cationic

eactants in Nafion film and consequently enhance its applica-ion in designing reproducible and ultra-sensitive electrochemicalensors. The unique properties of gold nanoparticles, highly dis-ersive 4-nitrophenylazo functionalised graphene (G/NPA) andafion 117 were combined in constructing a ternary nanocompos-

te system for improving the electroactivity of Nafion-incorporatedationic reactants. To demonstrate this, gold nanoparticle dot-ed 4-nitrophenylazo functionalised graphene nanocompositeAuNp/G/NPA) was systematically synthesised, immobilised on thelectrode surface using Nafion 117 and incorporated large amountf [Ru(bpy)3]2+ as a cationic reactant via electrostatic and ion-xchange interactions.

. Experimental

.1. Chemicals and reagents

Gold (II) chloride trihydrate (HAuCl4·3H2O), potassium per-anganate (KMnO4), sulfuric acid (H2SO4), tetrabutylammonium

etrafluoroborate (NBu4BF4), sodium tetrafluoroborate (NaBF4),-nitroaniline (C6H6N2O2), sodium nitrite (NaNO2), acetonitrileCH3CN, HPLC grade), hydrochloric acid (HCl, 37%), hydrogen per-xide (H2O2), hydrazine monohydrate (N2H4·H2O), diethyl ether,eagent grade ethanol, reagent grade methanol, sodium citrate,otassium phosphate dibasic (K2HPO4), hexamine rutheniumhloride (HRC), phosphoric acid (H3PO4) potassium phosphateonobasic (KH2PO4), potassium chloride (KCl), tris (bipyridine)

uthenium (II) chloride (Ru(bpy)3Cl2) were all supplied by Sigmaldrich, Nafion 117 solution was a Fluka product and naturalraphite powder was purchased from Alfa Aesar. The water usedhroughout all experiments was purified through a Millipore sys-em.

.2. Synthesis of highly dispersive 4-nitrophenyazo functionalisedraphene (G/NPA)

Graphene oxide (GO) was first prepared through a modifiedummer’s method using natural graphite powder as the start-

ng materials [13,21–24]. Briefly, 9:1 mixture of concentrated

Acta 128 (2014) 128–137 129

H2SO4/H3PO4 (180: 20 mL) was added to a mixture of graphitepowder (1.5 g) and KMnO4 (11.5 g) in a 250 mL capped round bot-tom flask. The reaction was then heated to 60 ◦C and stirred for12 h. The reaction mixture was cooled to room temperature andpoured onto ice that is mixed with 30% H2O2 in the ratio (400 mL:3 mL). The mixture was filtered and washed in succession with300 mL of 37% HCl solution in order to remove metal ions, 300 mLof ethanol, 300 mL of water until the rinse water pH became neu-tral. The solid obtained was vacuum-dried overnight at ambienttemperature [21].

G/NPA was synthesised from the as-synthesized graphene oxidein three steps by modified Yongchao Si and Edward T. Samulskimethod [16]:

1. Pre-reduction of GO: 10 mg of it was dispersed into separatesheets in 40 mL of distilled water contained in a 100 mL cappedflask by simple sonication. Then the pH was adjusted to 9–10using 10% w/v sodium carbonate solution. Then 1 mL of 1 MNaBH4 solution was added slowly with constant stirring. Themixture was allowed to stir at 80 ◦C for 1 h. This pre-reductionstep is necessary to achieve complete reduction in the final step(step 3) and to enable the functionalisation reaction in step 2 byincreasing the size of sp2-carbon domains for reaction with thearyl diazonium salt.

2. Functionalisations of pre-reduced GO: Functionalisation of thepre-reduced GO with 4-nitrophenyl diazonium tetrafluorobo-rate was done using the method of Saby et al. [25]. Aftercentrifuging and rinsing several times with distilled water, par-tially reduced GO was re-dispersed in 40 mL of distilled watervia mild sonication. 0.5 mg of the as synthesised 4-nitrophenyldiazonium salt was dissolved in 5 mL of 1:1 acetonitrile to watermixture and added to the GO dispersion, then stirred in ice bathfor 3 h.

3. Post-reduction with hydrazine: After centrifuging and rinsingwith distilled water severally, nitrophenylazo functionalised GOwas re-dispersed in 40 mL of distilled water. Then 3 mL of 1 Mhydrazine was added into the dispersion and the mixture waskept at 100 ◦C for 16 h, highly dispersive 4-nitrophenylazo func-tionalised graphene (G/NPA) was recovered by washing andcentrifuging severally with distilled water. It was dried in thevacuum oven at 70 ◦C for 3 h and kept in an amber bottle forfurther use.

The chemical reduction of the diazonium cation, leads to theelimination of a nitrogen molecule and the production of highlyreactive aryl radical. This radical attacked the pre-reduced GOsurface to form a covalent bond between the aryl group andthe GO. Simultaneously, the functionalised GO was reduced to 4-nitrophenylazo functionalised graphene with the elimination ofH2O [26–28].

2.3. Synthesis of gold nanoparticles-dotted 4-nitrophenylazofunctionalised graphene (AuNp/G/NPA)

The G/NPA was dispersed in 2 mL of 5 mM HAuCl4 solution bysonication for 5 min. This was then diluted to 20 mL with dou-

was no colour change. The sample was then cooled to room temper-ature, separated by centrifuge and washed five times with doublydistilled water. The resulting products were dried in a vacuum ovenat 70 ◦C for 3 h [29].

130 C.E. Sunday et al. / Electrochimica Acta 128 (2014) 128–137

ize go

2n(

tmwrtbwdcamPuu

h

Scheme 1. Schematic illustration on the preparation route to immobil

.4. Electrode preparation and modification using goldanoparticles-dotted 4-nitrophenylazo functionalised grapheneAuNp/G/NPA)

Prior to modification, glassy carbon electrode was first polishedo mirror-like surface with aqueous slurries of 1.0, 0.3 and 0.05

icro alumina powder and rinsed thoroughly with doubly distilledater between each polishing step. Residual polishing material was

emoved by washing successively with 1: 1 water to nitric acid solu-ion, absolute ethanol and doubly distilled water in an ultrasonicath, air dried and used immediately. The obtained AuNp/G/NPAas dispersed in 1:1 water to methanol solution to yield 1 mg/mLispersion by ultra-sonication for 1 h. Then, 2 �L of it was drop-asted on the pre-treated glassy carbon electrode and dried in

desiccator for 24 h at room temperature forming a thin filmodified glassy carbon electrode depicted as GCE/AuNp/G/NPA.

hosphate buffered (0.1 M) solution (PBS) of pH 7.2 was preparedsing 0.1 M K2HPO4, 0.1 M KH2PO4, 0.1 M KCl and kept at 4 ◦C before

se.

Scheme 1.The prepared PBS solution was used to prepare 5 mM

exaamineruthenium chloride solution (HRC). The relative

ld nanoparticles onto nitrophenylazo functionalized graphene surface.

electroactivity (Irel) of the modified electrode was quantified bycyclic voltammetry in the presence of 5 mM HRC electrolyte asredox probe. The parameter Irel is defined as:

Irel(%) = Ipa GCE/film/(Ipa Bare GCE)−1 × 100 (1)

Where Ipa is anodic peak current.

2.5. Preparation of sensor platform

Nafion stock solution was diluted with 1:1 water to methanolmixture to yield 1% (v/v) solution. AuNp/G/NPA was dispersedin the 1% (v/v) Nafion solution by ultra-sonication for 30 minto form 1 mg/mL uniform nanocomposite. 4 �L of it was drop-casted on the surface of a pre-cleaned glassy carbon electrode andthen excess solvent was allowed to evaporate to dryness in theopen at room temperature to form a thin film (the glassy car-bon electrode was cleaned following the steps described in section2.4 above). The Nafion/AuNp/G/NPA modified GC electrode was

placed in 1 mM Ru(bpy)3Cl2 aqueous solution for 3 h to incorpo-rate [Ru(bpy)3]2+ via electrostatic and ion-exchange interactions,forming a thin film modified glassy carbon electrode depicted asGCE/Nafion/AuNp/G/NPA/[Ru(bpy)3]2+.

himica Acta 128 (2014) 128–137 131

etmaecidiaic

2

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wwiwr0oatas

aimtpup

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3

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Fig. 1. (a) UV-visible spectra of gold nanoparticle, graphene oxide, 4 nitropheny-

C.E. Sunday et al. / Electroc

Large amount of the cationic signal probe [Ru(bpy)3]2+ werentrapped on the modified electrode due to the enlarged elec-roactive surface area of AuNp/G/NPA immobilized in the anionic

atrix of the perfluorosulfonate ionomer. Also the slight neg-tive charge conferred on the nanocomposite by NPA unitslectrostatically enhanced the quantity and stability of theationic reactant. Another GC electrode was modified follow-ng the same protocol without AuNp/G/NPA nanocatalyst. It wasepicted as GCE/Nafion/[Ru(bpy)3]2+ and both were character-

zed comparatively. Previous research has shown that Nafion as perfluorosulfonate polymer with a micellar pore structure canncorporate other molecules into its bulk membrane throughoulombic and/or hydrophobic interactions [17–19].

.6. Photochemical and physical characterizations

The particle size and mono-dispersity were determined byigh resolution transmission electron microscopy (HRTEM) usingTEM, S-2400 N, HITACHI, Japan). The samples for HRTEM charac-erization were prepared by placing a drop of the dilute sampleolution on a carbon-coated copper grid and dried at room tem-erature before measurements. The topography of AuNp/G/NPAanocomposite was verified by scanning electron microscopySEM, JEM-2100, JEOL, Japan) using an accelerating voltage of0 kV. Atomic force microscopy (AFM) was used to study the sur-ace reactivity in ambient conditions using Veeco NanoMan V

odel (Cambridge, USA), the samples were scanned with a sili-on tip at a spring constant of 1–5 N/m and resonance frequencyf 60 – 100 kHz. Samples were prepared by drop coating 10 �L ofuNp/G/NPA and G/NPA suspensions on glass slides followed byrying in air at room temperature to form a homogenous coverageefore measurements.

.7. Electrochemical measurements

The electrochemical behaviour of the designed sensor platformas investigated by cyclic voltammetry (CV); Osteryoung square-ave voltammetry (SWV); Chronocuolometry and Electrochemical

mpedance spectroscopy (EIS) using BASi 100B electrochemicalork station (LG Fayette) and Zhanna electrochemical work station

espectively. The measurements were carried out in a quiescent.1 M phosphate buffer saline solution of pH 7.2 with the scan ratef 50 mV/s in a conventional three-electrode cell at room temper-ture, using glassy carbon electrode (surface area = 0.071 cm2) ashe working electrode, a platinum wire as counter electrode andll the potentials mentioned in all experimental were referred totandard Ag/AgCl (saturated KCl solution).

10 mL of PBS solution was measured into electrochemical cellnd analytical grade argon from Afrox, South Africa was usedn de-aerating the solution for at least 10 min before measure-

ents. Argon atmospheric head space was maintained throughouthe duration of the experiment. The CV was recorded in theotential between 650 mV and 1350 mV. EIS measurements wassed to study changes in interfacial parameters at 0 mV a biasotential and the frequency range was 100 MHz–10 kHz.

. Results and discussion

.1. Photo-physical properties

The Uv–Vis spectrum of GO in Fig. 1 (a) above shows an absorp-

ion peak at 230 nm, which is consistent with those in the literature30–32]. This peak corresponds to �-�* transitions of aromatic C = Cond. After formation of graphene through the post-reduction ofhe 4-nitrophenylazo functionalised GO, the peak is observed at

lazo functionalised graphene (G/NPA), gold nanoparticles-dotted 4-nitrophenylazofunctionalised graphene (AuNp/G/NPA) and (b) Raman spectra of graphene oxideand nitrophenylazo functionalised graphene (G/NPA).

260 nm. The red shift from 230 nm to 260 nm suggests that theelectronic conjugation within graphene sheets was restored. Char-acteristic absorption peak for gold nanoparticle was observed at530 nm corresponding to the surface plasmon absorption of Aunanoparticles [33,34]. Noticeably, after the chemical reduction ofgold ions in the functionalised graphene, the Uv-Vis absorptionspectroscopy neither showed any absorption peak at 530 nm nor at260 nm. This quenching of the surface plasmon absorption peaks ismost probably attributable to the decrease in electron density dueto the charge transfer from Au nanoparticles to G/NPA [35]. Indi-cating that about 99% of the synthesized gold nanoparticles werestrongly coated on the functionalised G/NPA surface [29,36].

Raman spectra of the nanocomposite were examined to fur-ther prove the reduction of the functionalised graphene oxideto graphene in the post- reaction step. D band and G band inRaman spectra are usually utilized to characterize the reductionof graphene oxide. The shift of G band to lower wave number andthe increase of ID/IG ratio (peak intensity of D band: peak intensityof G band) are two major indicators for graphene oxide reduction.As exhibited in Fig. 1 (b), G band of graphene oxide shifts from1599 cm−1 to 1587 cm−1 and ID/IG ratio increases from 1.00 to 1.36.

This agrees well with most reports on graphene oxide reduction[30,37–39]. The cooperation between D and G peaks gives rise to aD + G combination band induced by disorder at about 2930 cm−1;however the observed low strength of the D + G peak in the

132 C.E. Sunday et al. / Electrochimica Acta 128 (2014) 128–137

Fig. 2. The HRTEM images of (a) AuNp/G/NPA, (b) G/NPA and SEM images of (c) AuNp/G/NPA, (d) G/NPA.

Fig. 3. 3-D AFM images of (a) AuNp/G/NPA (b) G/NPA and 2-D AFM images of (c) AuNp/G/NPA (d) G/NPA.

himica Acta 128 (2014) 128–137 133

sscwTg

n(tmcgdTohacapbs

Fgaiisci

Antc

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Go

C.E. Sunday et al. / Electroc

pectra indicates a very small disorder in the sample. The 2D bandhape and the high energy second-order 2D band at 2675 cm−1 indi-ate single layer structure and an increase in thickness of G/NPA,hich is attributed to the attachment of 4-nitrophenyl azo units.

he results of Uv–Vis and Raman spectra confirm the reduction ofraphene oxide to graphene in the post-reduction step (section 2.2).

The morphology and surface reactivity of the goldanoparticles-dotted 4-nitrophenylazo functionalised grapheneAuNp/G/NPA) was confirmed by examining its high resolutionransmission electron microscopy (HRTEM), scanning electron

icroscopy (SEM) and atomic force microscopy (AFM) imagesomparatively with that of 4-nitrophenylazo functionalisedraphene (G/NPA). The HRTEM image in Fig. 2 (a) clearly showsark spots as opposed to what is observed for G/NPA Fig. 2 (b).he presence of such spots can be attributed to the attachmentf gold nanoparticles on to G/NPA surface. Similar dark spotsave been observed by other authors with both pristine graphenend functionalised graphene as described elsewhere [29,37,40]. Itould be observed in Fig. 2 (a) that gold nanoparticles appeareds dark dots on a light shaded graphene substrate with averagearticle size of 30 nm. The G/NPA surface was uniformly decoratedy the Au nanoparticles with very few aggregations, indicating atrong interaction between the G/NPA and gold nanoparticles.

A monolayer graphene sheet could be observed clearly inig. 2 (b), indicating a smooth surface of the chemically reducedraphene single layer. This corroborates the fact that there was noggregation of graphitic sheets after post-reduction step. Indicat-ng the presence of negatively charged NPA units which in turn,mparted sufficient electrostatic repulsion to keep graphene sheetseparated in solution [16]. The SEM images in Fig. 2 (c)–(d) furtheronfirms that gold nanoparticles were homogeneously embeddednto the graphene lattice.

The measured 3-D and 2-D AFM surface morphology of theuNp/G/NPA nano-sheets shows a lot of small dots on the grapheneano-sheet suggesting successful incorporation of gold nanopar-icles with the graphene and an enlarged surface reactivity areaompared to that of G/NPA in Fig. 3 (a)–(d).

.2. Electrochemical properties

We have used hexaamineruthenium (III) chlorideRu(NH3)6]2+/3+ complex as cationic redox probe (its electro-hemistry is well known with one electron couple) to evaluatehe electroactivity property of the synthesised gold nanoparticles-otted 4-nitrophenylazo functionalised graphene (AuNp/G/NPA)n glassy carbon electrode surface. Fig. 4 (a) shows cyclic voltam-ogram recorded in aqueous 5 mM [Ru(NH3)6]2+/3+ in 0.1 M PBS of

H 7.2, at a scan rate of 50 mV/s for (i) AuNp/G/NPA modified glassyarbon electrodes and (ii) bare GC electrode. The voltammogramf the [Ru(NH3)6]2+/3+ cationic redox system is reversible withare GC electrode having anodic and cathodic peaks (Ep,a = 160 mV,

p,a = 56 �A) and (Ep,c = -247 mV, Ip,c = -73.1 �A) respectively. TheuNp/G/NPA modified electrode (GCE/AuNp/G/NPA) presented auasi-reversible electrochemistry with almost equivalent anodiceak current and peak potential with the CV of bare GCE, indicatinglmost 100% relative electro activity when substituted into equa-ion (1) in section 2.4. The slight reduction in cathodic peak currents due to inhibition of electron flow through GCE/AuNp/G/NPA

hen the electrode potential became more negative. The inhibi-ion effect is caused by the slight negative charge conferred onhe AuNp/G/NPA composite by NPA units. Consequently all thexidised species ([Ru(NH3)6]3+) in the vicinity of the electrode

here not reduced back to [Ru(NH3)6]2+.

From Fig. 4 (b), the insert shows the cyclic voltammogram of theCE/Nafion electrode in argon-degassed PBS of pH 7.2, at a scan ratef 50 mV/s. It did not exhibit any electrochemistry as expected since

Fig. 4. (a) CV in argon degassed 5 mM HRC in 0.1 M PBS of pH 7.2 at 50 mV/s of(i) GCE/AuNp/G/NPA (ii) Bare GCE. (b) CV in argon degassed 0.1 M PBS of pH 7.2 at50 mV/s of GCE/Nafion/[Ru(bpy)3]2+ and for GCE/Nafion in the insert.

Nafion is not electro-active but can incorporate other moleculesinto its bulk membrane through coulombic and/or hydrophobicinteraction [17,19,20] This property was explored as describedin section 2.5 to incorporate [Ru(bpy)3]2+ molecules to obtain amodified GC electrode depicted as GCE/Nafion/[Ru(bpy)3]2+. A welldefined redox couple could be observed at 1124 mV and 1026 mVfor its CV in argon degassed 0.1 M PBS of pH 7.2 at 50 mV/s.The redox couple consist of cathodic peak (Ep,c = 1026 mV, Ip,c = -5.9037 �A) and an anodic peak (Ep,a = 1124 mV, Ip,a = 7.1879 �A)with a midpoint potential E0, of 1075 mV. These peaks are due tooxidation of Ru2+ and reduction of Ru3+ respectively correspondingto the following redox system:

Ru2+ − 1e-� Ru3+

The synthesised gold nanoparticles-dotted 4-nitrophenyl azofunctionalised graphene (AuNp/G/NPA) was also used to design aelectrochemical sensor platform as described in section 2.5 and itselectrochemistry were compared with GCE/Nafion/[Ru(bpy)3]2+.

Fig. 5 (a) (ii) shows the electrochemistry ofGCE/Nafion/AuNp/G/NPA/[Ru(bpy)3]2+ indicating cathodic peak(Ep,c = 1032 mV, Ip,c = -7.0211 �A) and anodic peak (Ep,a =1126 mV,Ip,a = 9.3457 �A) with a midpoint potential E0, of 1079 mV. And

134 C.E. Sunday et al. / Electrochimica Acta 128 (2014) 128–137

Fig. 5. (a) CV’s performed in argon-degassed 0.1 M PBS, pH 7.2 at 50 mV/s for (i) GCE/Nafion/[Ru(bpy)3]2+ and (ii) GCE/Nafion/AuNp/G/NPA/[Ru(bpy)3]2+. (b) SWV’s of (i)G( S, pH

Fiaiccstsob

Giwroaupits

CE/Nafion/[Ru(bpy)3]2+ and (ii) GCE/Nafion/AuNp/G/NPA/[Ru(bpy)3]2+.c) Scan rate dependence CV’s of GCE/Nafion/AuNp/G/NPA/[Ru(bpy)3]2+ in 0.1 M PB

ig. 5 (a) (i) shows the electrochemistry of GCE/Nafion/[Ru(bpy)3]2+

ndicating cathodic peak (Ep,c =1026 mV, Ip,c = -5.9037 �A) andnodic peak (Ep,a = 1124 mV, Ip,a = 7.1879 �A). About 30% increasen the magnitude of the Ip,c and Ip,a could be observed in theyclic voltammogram of GCE/Nafion/AuNp/G/NPA/[Ru(bpy)3]2+

ompared to that of GCE/Nafion/[Ru(bpy)3]2+. The Osteryoungquare-wave voltammogram in Fig. 5 (b) (i) and (ii) corroboratehis finding. This result is attributed to the enlarged electroactiveurface area of AuNp/G/NPA which increased the loaded quantityf the signal probe under the electrochemical condition andenefits its contact with the transducer.

From the scan rate dependence voltammogram ofCE/Nafion/AuNp/G/NPA/[Ru(bpy)3]2+ in 0.1 M PBS of pH 7.2

n Fig. 5 (c), it was observed that the anodic peak potentials shiftsith increasing scan rate while the cathodic peak potentials are

elatively independent of varying scan rate, indicating the presencef surface bound electro active species. Notably, the ratio of thenodic to cathodic peak currents (Ia/Ic) for all scan rates is notnity (unsymmetrical) meaning that the redox kinetics for the two

rocesses are not similar. Therefore the electron transfer reaction

s quasi-reversible. The peak current (Ip) increased linearly withhe square root of the scan rate (v1/2) at low scan rates and deviatedignificantly at higher scan rates (plot not shown). This implies

7.2. (d) Plots of the dependence of cathodic peak current on scan rate.

that the charge transfer at the electrode-film interface, in theabsence of oxygen is diffusion controlled and also confirms thatthe electron transfer reaction for this system is quasi-reversible[41].

A plot of the log of cathodic peak currents against log of scan ratein Fig. 5 (d) gave a slope with a value that is approximately 1.0, indi-cating that the peak is an adsorption peak in which a surface-boundelectroactive species undergoes fast electron transfer reaction atthe electrode [42]. It also confirms a thin layer electrochemistry inwhich all the [Ru(bpy)3]2+ molecules in the film are converted fromRu2+ to Ru3+. The number of electrons transferred (n = 0.766) wasestimated using double potential chronocoulometry [41,43,44].This implies that the thin film was conducting and exhibited quasi-reversible electrochemistry in PBS solution involving one electronprocess.

The diffusion coefficient of solution species and sur-face concentration of the adsorbed [Ru(bpy)3]2+ for bothGCE/Nafion/AuG/NPA/[Ru(bpy)3]2+ and GCE/Nafion/[Ru(bpy)3]2+

were concomitantly determined by chronocoulometry [44]. The

electrolysis of solution species is diffusion-controlled, and dependson t½. In contrast, the electrolysis of adsorbed species is essentiallyinstantaneously, as well as the double layer charging. The equationfor the total charge Q is therefore given by:

C.E. Sunday et al. / Electrochimica

Fd

Q

o

Q

wQl(AFep

TeofoFio

ig. 6. (a) Chronocoulogram of GCE/Nafion/AuNp/G/NPA/[Ru(bpy)3]2+ in argon-egassed 0.1 M PBS, pH 7.2. (b) Anson plot of the data from (a).

= Qdiff + Qads + Qdl

r

= 2nFACD1/2 �−1/2 t1/2 + nFA� ∗ +Qdl (2)

here: Qdiff = charge due to electrolysis of solution species;ads = charge due to electrolysis of adsorbed species; Qdl = double-

ayer charge; �* = surface concentration of adsorbed speciesmol/cm2); D = diffusion coefficient of solution species (cm2/s);

= Surface area of the glassy carbon electrode, 0.071 cm2; = Faraday’s constant, 95484.56 C/mol; C = Concentration of thelectrolyte, 0.1 M; t = Time of potential step (s); T = time followingotential step (s)

The intercept of Q versus t½ plot is the sum of Qdl and Qads.he Qdl was eliminated from the equation by running identicalxperiment on the electrolyte alone. Noticeably, the adsorptionf [Ru(bpy)3]2+ specie produced significant changes in the inter-acial capacitance, therefore values of Qdl evaluated in the absence

f the reactant did not apply when the reactant is present. Fromig. 6 (b), the intercept value is 1.27178E-7. Applying this valuen nFA�*, the surface concentration (�*) of adsorbed [Ru(bpy)3]2+

n GCE/Nafion/AuNp/G/NPA/[Ru(bpy)3]2+ was calculated to be

Acta 128 (2014) 128–137 135

1.87 × 10−11 mol/cm2 while the same experiment repeated forGCE/Nafion/[Ru(bpy)3]2+ gave �* value of 1.22 × 10−11 mol/cm2

which is about 34.76% lower.Again this result is attributed to the large specific surface

area of gold nanoparticle dotted 4 nitro-phenyl azo functionalisedgraphene (AuNp/G/NPA) which increased the loaded amount of[Ru(bpy)3]3+. In addition, the slight negative charge conferred onthe nanocomposite by NPA units enhanced the quantity and sta-bility of the cation electrostatically. The diffusion coefficient forGCE/Nafion/AuNp/G/NPA/[Ru(bpy)3]2+ from the slope (2nFACD½

�-½) of Q versus t½ Anson plot was calculated as 1.12 × 10−18

cm2/s. About 25% decrease in diffusion coefficient was observedfor GCE/Nafion/[Ru(bpy)3]2+.

The Nyquist plot of two electrode systems [GCE/Nafion/[Ru(bpy)3]2+ and GCE/Nafion/AuNp/G/NPA/[Ru(bpy)3]2+] at 0 mVare shown in Fig. 7 (a). The Nyquist plot is based on an equivalentcircuit Figure 8 (b) in which the solution resistance, Rs, precedes aconstant phase element, CPE, which is in parallel with charge trans-fer resistance, Rct and warburg impedance, (Zw). Rct representsthe resistance to charge transfer kinetics between the electrolyteand the electrode. The Rct and Rs of GCE/Nafion/[Ru(bpy)3]2+

and GCE/Nafion/AuNp/G/NPA/[Ru(bpy)3]2+ are (Rct = 1955 �;Rs = 199.4 �) and (Rct = 939.3 �; Rs = 74.51 �) respectively.This shows a 51% less in Rct and 62.6% less in Rs values ofGCE/Nafion/Ru(bpy)3

2+ compared to GCE/Nafion/AuNp/G/NPA/[Ru(bpy)3]2+. The reduction in Rct value is attributed tolarger amount of the cationic reactant, [Ru(bpy)3]2+, that wasloaded on GCE/Nafion/AuNp/G/NPA/[Ru(bpy)3]2+ as confirmed bychronocoulometric studies above.

This in turn benefits closer contact between the signal probeand the transducer and consequently faster electron transfer reac-tion. The Rs value represents the property of the bulk solution thatreflects uncompensated Ohmic resistance via non-Faradaic process[45]. The drop in Rct value of GCE/Nafion/AuNp/G/NPA/[Ru(bpy)3]2+

led to faster reduction of ions in proximity of the electrode caus-ing higher concentration gradient in the bulk electrolyte solution.Therefore oxidised ions in solution diffuse faster towards the elec-trode surface as the solution resistance is reduced. A comparativestudy of the interfacial heterogeneous electron transfer rate of bothelectrodes was done using equation 3 and 4 [41].

Rct = RT/nFIo (3)

Io = nFAk0C∗ (4)

Where n (number of electrons transferred) = 1 e, F (Fara-day constant) = 95484.56 C/mol, R (Gas constant) = 8.314 J/mol/K, T(Reaction temperature) = 298 K, A (Geometric area of elec-trode) = 0.071 cm2, C* (concentration of PBS electrolyte) = 0.1 M, Io(Standard exchange current) and k0 (Heterogeneous rate transferconstant cm/s).

The values of Io for GCE/Nafion/[Ru(bpy)3]2+ and GCE/Nafion/AuNp/G/NPA/[Ru(bpy)3]2+ were 1.33 × 10−5 A and2.76 × 10−5 A, respectively. And the corresponding k0 valueswere 1.9 × 10−8 cm/s and 4.0 × 10−8 cm/s. The larger k0 value forGCE/Nafion/AuNp/G/NPA/[Ru(bpy)3]2+ confirms that the film hasfaster charge transfer reaction than GCE/Nafion/[Ru(bpy)3]2+.

The frequency dependence of both the impedance and thephase angle is shown in the Bode plots of Fig. 7 (c) and (d). Theimpedance-dependence plots (Figure 8 (c)) shows a decreasein the impedance of the electrodes as the frequency increases.However, the impedance of Nafion/AuNp/G/NPA/[Ru(bpy)3]2+

modified electrode was lower than that of Nafion/[Ru(bpy3)]2+

modified electrode over the frequency range studied, whichindicates improved conductivity of AuNp/G/NPA containingsystem. The phase shift of both electrodes where below 45o atlowest frequency in Fig. 7 (d). However, the phase shift increased

136 C.E. Sunday et al. / Electrochimica Acta 128 (2014) 128–137

F nd (dG s pote

waav[dote[

4

ipgce13tet

ig. 7. (a) Nyquist plots, (b) circuit diagram, (c) total impedance Bode plots aCE/Nafion/[Ru(bpy)3]2+, measured in argon-degassed 0.1 M PBS, pH 7.2 at zero bia

ith frequency for GCE/Nafion/AuNp/G/NPA/[Ru(bpy)3]2+ until characteristic frequency, fc = 2.4 Hz, where the phase shift has maximum value of 65.7◦, and then it decreased to lowestalue of 4.5◦ which is expected for simple equivalent circuit46]. On the contrary, the phase shift of GCE/Nafion/[Ru(bpy3)]2+

ecreased constantly with higher frequencies to lowest valuef 4.5◦. This implies that the modification of the GC elec-rode with Nafion/AuNp/G/NPA/[Ru(bpy)3]2+ improved thelectro-activity of the electrode surface better than Nafion/Ru(bpy3)]2+.

. Conclusions

Photo-physical characterizations of AuNp/G/NPA/[Ru(bpy)3]2+

ndicated that gold nanoparticles were homogeneously incor-orated onto highly dispersive 4-nitrophenyl azo functionalizedraphene. The AuNp/G/NPA nanocomposite improved the electro-atalytic efficiency of [Ru(bpy)3]2+/3+ in the nafion film. Thelectrochemical sensitivity of AuNp/G/NPA was reflected by its00% relative electro-activity, 30% increase in peak currents and

5% reduction in Rct compared with pure nafion. This is attributableo the increase in the quantity of [Ru(bpy)3]2+ loaded on thenlarged electro-active surface area of the nano-composite elec-rode. The slight negative charge conferred on the nanocomposite

) phase angle Bode plots for (i) GCE/Nafion/AuNp/G/NPA/[Ru(bpy)3]2+ and (ii)ntial, 5 mV alternative voltage and frequency range of 100 MHz–10 kHz.

by NPA units also contributed in enhancing the quantity and sta-bility of the cation electrostatically. It is therefore certain thatthe sensitivity and stability of the cationic reactant, [Ru(bpy)3]2+,was improved by its incorporation with GCE/Nafion/AuNp/G/NPAplatform. This study showed that Nafion/AuNp/G/NPA com-posite may be used to develop a more reproducible, high-performance, ultra-sensitive electrochemiluminescence, voltam-metric, impedimetric and amperometric sensors employing[Ru(bpy)3]2+ as the electroactive species or any other cationicreactant.

Acknowledgement

The study was made possible by research grant and PhD bur-saries from Mintek Nanotechnology Innovation Centre (NIC) andthe National Research Foundation (NRF) of South Africa.

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