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Electrochimica Acta 114 (2013) 590– 597

Contents lists available at ScienceDirect

Electrochimica Acta

jou rn al hom ep age: www.elsev ier .com/ locate /e lec tac ta

lectrochemically reduced graphene–gold nano particle composite onndium tin oxide for label free immuno sensing of estradiol

enkataraman Dharumana,b,∗, Jong Hoon Hahnb,∗∗, Kumarasamy Jayakumara, Wei Tengb

Molecular Electronics Laboratory, Department of Bioelectronics and Biosensors, Science Block, Alagappa University, Karaikudi 630 004, IndiaDepartment of Chemistry and BioNanotechnology Center, Pohang University of Science and Technology, San 31, Hyojadong, Pohang 790-784, South Korea

r t i c l e i n f o

rticle history:eceived 21 August 2013eceived in revised form 11 October 2013ccepted 15 October 2013vailable online 29 October 2013

eywords:raphenelectrochemicaluNPabel freestradiolmmunosensing

a b s t r a c t

Electro reduced graphene and gold nano particle (ErG/AuNP) composite is prepared on indium tin oxide(ITO) surface. Characterization by X-ray diffraction (XRD), field emission scanning electron microscope(FESEM), Raman spectroscopy (RS), atomic force microscopy (AFM), X-ray photoelectron spectroscopy(XPS) and transmission electron microscopy (TEM) techniques reveals the formation of vertical and flatoriented ErG films on the ITO. The AuNP deposition changes the flat oriented ErGs into vertical orienta-tion indicated by the FESEM. Coherent interactions between the ITO, ErG and AuNPs are responsible forthe discrete formation of vertical oriented hetero structures of ErG–AuNP composite on the ITO. Electro-chemical properties are investigated using [Fe(CN)6]3−/4− and [Ru(NH3)]2+/3+ redox probes using cyclicvoltammetry (CV). While the [Fe(CN)6]3−/4− shows fast reversible behavior, the [Ru(NH3)]2+/3+ revealsvery slow charge transport on both ErG and ErG/AuNP films indicating the multi and compact graphenelayer posses positive charge at pH 6.5 used for preparing these composites. Immuno sensing of breastcancer inducing hormone 17�-estradiol (E2) is demonstrated in presence of [Fe(CN)6]3−/4−. Estrone (E1)and estriol (E3) antigens are used as the controls. The near vertical immobilization of anti-estradiol-

−3 −12

antibody enhances the lowest detection limit of 0.1 fmol and dynamic range of 1 × 10 –0.1 × 10 Mwithout any signal amplifiers. These results prove that the acid group of the GO is reduced selectivelyin controlled way by simple potential cycling method which could not be achieved by other chemicalreduction methods. Hence, the protocol is an alternative, simple and inexpensive technique for produc-ing few layered vertical graphene and ErG/AuNP composite than the solution based chemical synthesismethods reported.

. Introduction

Interaction of 17�-estradiol [1–4] with tumor suppressor geneRCA1 and p53 is essential to maintain the genetic process stabilitynd to prevent the risk of breast cancer. Concentration of 17�-stradiol greater than picogram in blood and urine samples of postenopausal women affects the genetic cycle and promotes prolif-

ration of breast cancer cells. Because of this hormonal dependency,hromatography [5–8], mass spectrometry [7] and electrochemi-

al [9–27] methods have been reported for estrogen estimations.lthough electrochemical method is simple, direct, amenable

or portable and hand held device production (similar to the

∗ Corresponding author at: Molecular Electronics Laboratory, Department of Bio-lectronics and Biosensors, Science Block, Alagappa University, Karaikudi 630 004,ndia. Tel.: +91 4565 226385; fax: +91 4565 225202.∗∗ Corresponding author. Tel.: +82 54 2792118; fax: +82 54 2795805.

E-mail addresses: dharumanudhay@yahoo.com (V. Dharuman),ahn@postech.edu (J.H. Hahn).

013-4686/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.electacta.2013.10.128

© 2013 Elsevier Ltd. All rights reserved.

commercial gluco meters), careful designing of the sensor (trans-ducer) surface is essential to achieve high selectivity andsensitivity of the target analyte sensing. Applications of elec-trodes like mercury [9,11], platinum nano particle [14], carbon[16,17,14,18–22,12,23] and molecularly imprinted Pt/graphene[24] are reported for estrogen sensing, but similar oxidation poten-tials of different estrogens limit the selectivity and impede practicalapplications. This has been overcome by developing highly selec-tive DNA [28–32] and immuno [24,27,33–37] sensors. In thecontext of electrochemical immuno sensing, enzymes (horse radishperoxidase [24,27,33] and alkaline posphatase [35–37]) and metalnano (gold and silver [38,39]) particles are used for signal amplifi-cation.

.1 Label free electrochemical method that uses unlabeled targetsis emerging as an alternative and cost effective method [40–53]in the field of biosensor development compared to the exist-

ing fluorescence and chromatographic techniques. Carbon nanostructures including graphene are being investigated for vari-ous label free biosensing [50–53] applications. Since electronicproperties of the graphene and graphene–metal nano particle

V. Dharuman et al. / Electrochimica Acta 114 (2013) 590– 597 591

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Scheme 1. Fabrication of ErG/AuNP

omposites depend on their preparation origin, chemical vapoureposition (CVD) [54] and electrophoretic [55,56] methods haveeen reported for preparing graphene based surfaces for vari-us applications. Bio molecules (e.g., DNA and antibodies) aremmobilized directly on the functional groups of graphene oxideGO), however, the literature survey suggests that the directlymmobilized proteins layers on the carbon surfaces are unstable57,58]. Haque et al. have used monolayer of ErG film fabri-ated on the ITO surface for the immuno sensing of estradiol27], but the HRP enzyme labeled target antigen is used for sig-al amplification. Till now no report available on the label free

mmuno sensing of the estradiol using graphene transducers inhe literature. Hence, this report aims to construct graphene–AuNPomposite on the ITO surface for the label free immuno sensingf 17�-estradiol (E2) selectively in presence of similar structuredstrone (E1) and estriol (E3), Scheme 1. The ErG film and AuNPsre directly anchored on the ITO surface by sequential electroeduction using CV technique to create ITO/ErG and ITO/ErG/AuNPurfaces. The surfaces are characterized using RS, FESEM, AFM, XPSnd TEM. The immuno sensing of estradiol is made on both theTO/ErG and ITO/ErG/AuNP in presence of [Fe(CN)6]3−/4− in phos-hate buffer. The sensor showed high selectivity for the estradiolith lowest detection limit of 0.1 fmol without the HRP enzyme

abels.

.1. Experimental

.1.1. ReagentsEstrone (E1), estradiol (E2) and estriol (E3) were purchased

rom Sigma–Aldrich (USA). The anti estradiol antibody from rab-it (1 mg mL−1) was purchased from Fitzgerald, USA. Graphiteowder (50 nm) was purchased from Loba chemie Pvt Ltd, Mum-ai, India. Sulphuric acid, potassium permanganate, hydrogeneroxide, sodium chloride, ethylene diamine, methylacrylate,ilver nitrate, sodium borohydrate, HAuCl4·3H2O, K3[Fe(CN)6],4[Fe(CN)6], NaH2PO4, NaOH, KOH, KCl, 3-mercapto-propinoc acid

MPA), trisodium citrate, 1-ethyl-3-(3-dimethylaminopropyl) car-odiimide) hydrochloride (EDC) and N-hydroxysuccinimide (NHS)

ere all obtained from Sigma–Aldrich, USA. De-ionized water (DI)as used to prepare all experimental solutions. ITO was purchased

rom Sigma–Aldrich and laser cut into chips of physical dimension.2 cm × 1.5 cm.

O for immune sensing of estradiol.

1.2. Instruments

Three-electrode cell consisting of Ag/AgCl reference, Pt wirecounter and ITO working electrodes was used for all electro-chemical measurements. Area of the working electrode calculatedfrom the inner diameter of the O-ring was 0.196 cm2. CV datawere acquired using VMP multi channel potentiostat (PerkinElmerInstruments Boston, MA). Scanning electron microscope (SEM)images and energy dispersive spectrums (EDSs) were recordedwith JSM-5900 (JEOL, Japan) instrument. Atomic force microscopyimages in tapping mode were obtained with multi-mode atomicforce microscope from Veeco Instruments Inc., USA. Raman spec-tra were recorded using WiTech H [c] Alpha 300 which uses theexcitation wavelength 532.34 nm. XRD measurements were madeusing Bruker D8-Advance powder diffracto meter which uses CuK� (2.2 kW max). TEM images were acquired using Tecnai G2 20instrument from FEI Company, Hillsboro, USA. The XPS analysis wascarried out on Kratos-Axis Ultra XPS instrument with a hemispher-ical 165 nm electron energy analyzer using Al K� radiation.

1.3. Preparation of electro reduced graphene–gold nano particlelayer on indium titanim oxide surface and biofunctionalization

The ITO plate was cleaned successively in water, acetone,ethanol and water for 15 min. GO was prepared following the mod-ified Hummers method [59]. 1 mg mL−1 of as-synthesized GO wasdispersed in phosphate buffer (pH 6.5) and ultra sonicated for 3 h.The ITO electrode was potential scanned from 0 to −1.6 V (Ag/AgCl)continuously for 75 cycles at a scan rate 50 mVs−1 to form the ErGdirectly on the ITO surface. Following this, the ErG surface wasscanned in 0.5 mM HAuCl4·nH2O in phosphate buffer for 25 cyclesto deposit the AuNP. The ITO/ErG/AuNP surface was incubated inPBS buffer (pH 7.4) containing 1 mM MPA for 4 h. The acid group ofMPA was activated by immersing in the 5 mM EDC and NHS (1:1,v/v) solution for 3 h. Since direct immobilization of antibody on thegold nano particle results in improper orientation and low reac-tivity, covalent immobilization on the MPA has been followed inseveral reports. The surface was then interacted with rabbit anti-estradiol antibody for 12 h at 4 ◦C and washed with blank buffer

to remove the un-reacted antibodies. The free reactive sites on thesurface after antibody immobilization was then passivated using1% BSA for 2 h. 17�-estradiol antigen, which was previously dis-solved in ethanol and diluted using phosphate buffer to the required

592 V. Dharuman et al. / Electrochimica Acta 114 (2013) 590– 597

Fig. 1. FESEM images of ITO/ErG (A) and ITO/ErG/AuNP (B). Measuring scale 100 nm.

. (B) A

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Fig. 2. (A) AFM image of vertical ErGs on ITO

oncentration, was then allowed to react for 2 h, Scheme 1. Afterach step, the surface behavior was interrogated intermittentlysing the CV in presence of 1 mM [Fe(CN)6]3−/4− in phosphateuffer.

. Results and discussion

.1. Characterization of electro reduced graphene layer onndium titanim oxide surface and immuno sensing of estradiol

Fig. S1A shows the CV profile of the electro reduced grapheneErG) on the ITO plate (abbreviated as ITO/ErG film) from theOs dispersed in phosphate buffer by continuous potential cycling

eduction (75 cycles) method. Formation of the ErG is indicatedy the appearance of a reduction peak at −1.5 V. Fig. S1B com-ares the CV profiles of the ErG films formed during the first and

ast (75th) cycles used for reducing the GO functional groups. Twoedox pairs, P1 and P2, are appearing in the very first cycle in theotential region between −0.8 and −1.6 V. Observed peak poten-ials for the P1 and P2 peaks are −1.0, −0.89 and −1.35, −1.03 V,espectively. The multiple peaks may be due to the reduction of dif-erent functional groups, however, could not be assigned exactly atresent. Redox peak currents for the peaks at potentials ca. −1.35nd −0.89 V are increasing continuously till 20 cycles and thenecreasing. The other two peaks (appeared at −1.0 and −1.03 V)re completely absent from the second cycle onwards. In addi-ion, while the position of the reduction peak at −1.35 V shifting

oward higher negative potential (ca. Epc, −1.5 V) with increasingotential cycling number, the oxidation peak position remains con-tant at −0.89 V until the final cycle is completed. This is constantlybserved on all electrodes studied. The consistent appearance of a

FM image of vertical ErGs/AuNP on the ITO.

single redox pair after the final cycle suggests that the GO functionalgroups undergo reversible redox processes within the potentialwindow applied. No peeling of the ErG film observed while washingwith DI water after electro deposition suggesting its high stability.This observation is in conflict with the literature reports [27,60]wherein only one reduction peak is appeared and the peak is absentfrom the second cycle onwards. This signatures the complete reduc-tion of the fewer number of GO particles that are in direct contactwith the electrode surface during the first cycle itself and no reoxi-dation occurred. The FESEM, Fig. 1A, shows highly dense, irregularand rough morphology of the ErG film formed on the ITO surface.

The morphology is different from the transparent, uniform andflat graphene films reported by Seo et al. [61], Haque et al. [27] andYang et al. [60]. The morphological differences may arise from dif-ferent methods of GO reduction used. For instance, while Seo et al.[61] have used CVD thin film deposition and patterned by the ICP,Haque et al. [27] have used the potential cycling method to formthe ErG on the ITO. The constant potential electrophoretic reduc-tion method also showed thick and wrinkled structures, reportedby Srivastava et al. [56]. Hence, the contrast FESEM film morphologyobserved in the present work is due to the regular arrangement ofgraphene sheets and their tendency to orient vertically with respectto the flat ITO surface. This occurs in the following two steps. First,the ErGs initially formed during the first few cycles are depositedin a flat orientation continuously till 20 cycles. Second, as the num-ber of potential cycling increases, the flat oriented ErG films maytend to orient vertically to accommodate the additional ErGs being

formed, Scheme 1. This is clearly evidenced in the FESEM, Fig. 1A.The graphene layer is tilted away from 90◦ at some places com-pared to other areas where the graphene sheets are nearly standingupright positions on the flat ITO. The TEM in Fig. S2A reveals the

V. Dharuman et al. / Electrochimica Acta 114 (2013) 590– 597 593

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ig. 3. Raman spectra of ITO (curve a), ITO/ErG (curve b) and ITO/ErG/AuNP (curve).

nife edged graphene layers fabricated on the ITO. The verticalrientation is further confirmed by the AFM that showedubular like structure for the ITO/ErG surface, Fig. 2A with theverage height ∼400 nm. Literally, it is supported from the observa-ion of ribbon like AFM image for the graphene layers prepared by

icrowave plasma chemical vapor deposition (MPCVD) techniquen Si and Cu substrates [54,62–64]. Different experimental condi-ions such as the solution phase and potential cycling method usedor the growth of graphene films on the ITO surface are the respon-ible factors for the different structural features compared to thePCVD grown graphene films.The D and G bands are appeared at 1330 and 1520 cm−1, respec-

ively, in the RS of the ITO/ErG, Fig. 3, curve b, and the ID/IG ratios 0.87. The 2D band is broad and up-shifted to 2923 cm−1 thanhe sharp and high intense 2D band observed (2700 cm−1) forhe single layer graphene on Si. The peak in the frequency region00–500 cm−1 is due to the amorphous nature of the ITO beneathhe ErG film. While the D band appears from the disordered stackingf graphene layers, atomic defects and edges, the G band arises fromhe first order Raman scattering process for the in-plane vibrationf SP2 bonded carbon atoms.

The 2D band originates from a two phonon double resonanceaman process and closely related to the band structure of theraphene layers and does not require defects for its activation.

hese changes indicate the formation of graphene layers contain-ng more than five layers similar to graphite [62] with interlinkingetween them. The multi layer is revealed by the knife edged TEM

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Fig. 4. XPS spectra of ITO/ErG/AuNP.

Fig. 5. XRD pattern of ITO (curve a), ITO/ErG (curve b) and ITO/ErG/AuNP (curve c).

images shown in Fig. S2A. It must be recalled here that the CVDprepared graphene exhibited well defined D, G and 2D bands withIG/I2D ratio equal to 1 confirming the deposition of single layergraphene on the ITO substrate [27]. The D and G bands positions at1354 and 1520 cm−1 and D′ position at 1573 cm−1 (clearly visiblein enlarged scale) suggest the presence of structural disorders inthe ErG film prepared in the present work. In order to investigatethe nature of functional groups in the ErG film prepared, the XPSinvestigation is made. Fig. 4A shows C1s XPS spectrum of carbon.The presence of peak at 285.6 eV and its deconvolution into threesub peaks, centered at 285, 285.6 and 286.36 eV, indicate the pres-ence of SP2 carbon, ‘defect peak’ and C O groups. The absence oftailing peak between 286 and 290 eV in the C1s spectrum confirmsthe absence of acid functional carbonyl (C O) groups. The appear-ance of O1s peak, Fig. 4B, at 532.5 eV corresponding to the C OHbonds and is blue shifted by 0.4 eV compared to the 532.9 eV notedfor the MPCVD prepared FLGs on the Si substrate [54]. In contrast,the XPS C1s spectrum of as-synthesized GO showed C O peak inthe range from 286 to 290 eV, Fig. S3, for the presence of the COOHgroups.

The XRD pattern of the ITO (curve a) and ITO/ErG (curve b) arepresented in Fig. 5. Compared to the unmodified ITO that showeda broad peak below 30◦, the ITO/ErG surface showed four peaks forthe formation of graphene nano sheets. The absence of peak below2� 20◦ for the ITO/ErG surface (Fig. 5, curve b) suggests the absence

of unreduced GO on the ITO. The inter layer distance (0.39 nm), cal-culated from the (002) peak at 2� 32.86◦, is greater than 0.35 nm.The higher ‘d’ value suggests the presence of defects, oxygen

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594 V. Dharuman et al. / Electrochimica Acta 114 (2013) 590– 597

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ig. 6. (A) CV behaviors of ITO (curve a) modified with ErG (curve b) and AuNP (c0 mV s−1 in the potential window −0.6 to 0.7 V. (B) CV behaviors of ITO/ErG/AuNPntigen (curve f) measured under similar experimental conditions as in (A).

unctionalities and interlocked water molecules between theraphene sheets [47].

The electrochemical property of the ITO/ErG is investigated fur-her using the [Fe(CN)6]3−/4. The ITO/ErG surface gives increasedeak-to-peak separation of �Ep (Epa − Epc = 275 mV) and decreasedeak current (e.g., anodic peak current ipa, 22 �A, curve b) than thenmodified ITO surface (�Ep: 183 mV, ipa: 29 �A, curve a), Fig. 6A,

ndicating the restricted charge transfer between the ITO/ErG filmurface and [Fe(CN)6]3−/4−. Decreased charge transport is eitherue to the electrostatic repulsion between the negatively chargedpoxide and hydroxyl groups (which are partially reduced indi-ated by the XPS C1s spectrum, Fig. 4, similar to the XPS of the asynthesized GO, Fig. S3) of the ErG and [Fe(CN)6]3−/4− or the thickrG film may block the direct diffusion of [Fe(CN)6]3−/4− throughores to the unmodified ITO surface. In order to delineate theseffects, the redox property of the positively charged [Ru(NH3)6]2+/3+

n the ITO/ErG film is studied next.The redox current of the [Ru(NH3)6]2+/3+ is nearly absent, Fig. S4

inset) and hence, the ITO/ErG surface may effectively blocks directiffusion of both the redox probes through pores. The contrastehavior of these two probes at the ITO/ErG film suggests that therG film is positively charged at pH 7.4 [65] and hence, the electro-tatic repulsion between the graphene functional groups (epoxide,ydroxyl) and [Fe(CN)6]3−/4− is insignificant. This is supported formhe observation of decreased current for the [Fe(CN)6]3−/4− for thenreduced GO attached on the ITO through aminoethyl benzeneiazonium monolayer due to electrostatic repulsion between thenreduced COO− and [Fe(CN)6]3−/4−. This is uphold further by the

ncreased redox behavior of the [Fe(CN)6]3−/4− on the conductingrG film from by the electro reduction of the GO particles [27].hat is, the multi layered or regularly stacked graphene sheetsormed in this study are poor conductor of electricity. To concretehis effect further, the ErG film is prepared on the conventionallassy carbon electrode surface and electrochemical activities ofhe [Fe(CN)6]3−/4− and [Ru(NH3)6]2+/3+ are examined. The GCE/ErGlso showed the decreased charge transfer for Fe(CN)6]3−/4− andbsence of current for [Ru(NH3)6]2+/3+ probes, respectively, ingreement with the ITO (data not given).

For immuosensing of estradiol on the ITO/ErG film, the anti-stradiol-antibody has been immobilized covalently using theDC/NHS coupling agents followed by free surface blocking using

SA. The surface is then interacted with the estradiol antigeno form the antibody-antigen complex. However, no change inhe redox signal of the ITO/ErG, ITO/ErG-antibody and ITO/ErG-ntibody–antigen complexed surfaces is noticed in presence of the

) measured in presence of 1 mM [Fe(CN)6] in phosphate buffer at a scan rateified with MPA/EDC/NHS (curve d), anti-estradiol antibody (curve e) and estradiol

[Fe(CN)6]3−/4−. This is against the previous reports [27,54] whichused ITO/ErG and showed efficient discrimination of the antibodyand antibody-antigen complex formation. In other words, the anti-bodies are immobilized effectively on the COOH groups present inthe ErG films reported previously. Thus, comparison of our resultswith the literature results confirms the fact that the antibody isnot immobilized on the ITO/ErG film prepared in the present work.This corroborates the results from the XPS technique that indi-cated absence of acid functional groups (C1s XPS spectra, absence oftailing peak in the range 286–290 eV, Fig. 4). Collectively, the elec-tro activity and biosensing properties of the ErG films depend onthe preparation conditions employed such as the base substrate,constant potential electrolysis, continuous potential cycling andsolution pH.

2.2. Preparation and characterization of electro reducedgraphene and gold nano particle composite film on indiumtitanim oxide surface and immunosensing of estradiol

Since carbon–AuNP composites are highly sensitive, evidencedby the applications of carbon nano tubes and metal NPscomposites, the AuNPs are deposited on the ErG by the continuouspotential cycling (25 cycles) method to form ErG/AuNP compos-ite on the ITO. The Au deposition is indicated by the appearanceof reduction peaks at −0.2, −0.5 and −1.12 V, respectively, duringthe first cycle, Fig. S1C. However, only one reduction peak appearsat −0.5 V from the second cycle onwards. And the decrease ofpeak current with increasing number of potential cycles again evi-dences the poor conducting nature of the ErG film. The AuNPs aresparsely deposited and even agglomerated on the ITO/ErG surface,Fig. 1B. It is reported that the negatively charged AuNPs prefer-ably interact on the graphene surface due to the negative chargerepulsion from the pi electrons present on the edges of graphene[66]. In the present work, the AuNPs may penetrate through thefilm and attached on both the graphene functional groups (epoxide,hydroxyl) and the edges oriented at the junction point of indiumoxide and graphene layers. The vertical nature of the ErG film ismore pronounced in presence of the AuNPs as indicated by theAFM, Fig. 2B, and the AuNP deposition increases the graphene tubeheight ∼800 nm and roughness. The optical image also confirms thepresence of ErG/AuNP film on the ITO plate, Fig. S6A and B.

The graphene sheets on the ITO may undergo conformationalchange, stress, and hence, the film morphologies in presence ofAuNPs. The XRD pattern of the ITO/ErG/AuNP showed severaladditional peaks corresponding to (1 1 1), (2 0 0), (2 2 0), (3 1 1)

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reported 700 aM detection limit using impedance spectroscopy,but HRP enzyme labeled target antigen and hydroquinone areused for signal amplification. Kim et al. [67] have reported the

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nd (2 2 2) planes in addition to the (0 0 2) plane indicating theace centered cubic crystal structure of AuNP on the ErG film.owever, the AuNP deposition decreases the XRD peak intensity of

he (0 0 2) plane observed for the ITO/ErG surface. It may be notedhat the AuNP deposition at constant potential −0.25 V showedniform distribution of the AuNP on the ITO/ErG surface [64] andize of NPs dependent on the applied potential, time of electroly-is and strength of the interactions between the metal oxide andPs. The continuous reduction of AuNPs by the potential cyclingethod and their discrete distribution on the ITO/ErG film indi-

ates that the AuNP may differentially interact with the tin oxidearticles present in the indium-tin oxide surface (Fig. S5A) to form

arger and smaller AuNPs on the ITO/ErG, Figs. 1B and 2B. Theiscrete formation of ErG/AuNP may also arise from the changef flat oriented ErG to the vertical orientation during continu-us AuNP deposition. Therefore, the FESEM images of both theTO/ErG/AuNP and the unmodified ITO, Fig. S5A, show the nanoot like surface structures. It has been reported by Kou et al. [66]hat the ITO–graphene–PtNP composite prepared by the chemicaleduction method prevents agglomeration. To validate this on theTO/ErG surface, the ITO/ErG/PtNP surface is prepared under simi-ar experimental conditions and examined by CV, XRD and FESEM,ig. S7. The electronic properties are nearly same as that of theTO/ErG/AuNP. The XRD pattern of the ITO/ErG/PtNP is simpler andower intense than the ITO/ErG/AuNP pattern. The FESEM showedven distribution with minimal agglomeration of the PtNPs on theTO/ErG. The PtNP particles distribution and interaction with therG is completely different from that of the AuNP on the ITO asell.

The XPS, Fig. 3C, showed two distinct peaks for Au 4f5/2 andu 4f7/2, respectively, with an energy difference 3.9 eV inducedy different sized AuNPs. The TEM of the ErG/AuNP showed smallnd agglomerated AuNPs, Fig. S2B, and the average particle sizebserved is ≤5 nm, Fig. S2C. The RS pattern of the ITO/ErG/AuNPs shown in Fig. 3, curve c. Two observations are different fromhat of the ITO/ErG film. First, the appearance of high intense D, GD: 1354 and G: 1587 cm−1) along with 2D (2698 cm−1) and 2D′

2919 cm−1) bands with decreased ID/IG ratio 0.63 suggesting theormation of highly disordered graphene/AuNP film. The presencef 2D band strongly suggests the existence of highly turbo staticano graphite arranged in ordered stacking of graphene layers,cheme 1. The high intensity peak prominently observed below000 cm−1 for the ITO/ErG is decreased extensively in the presencef AuNP. The AuNP deposition enhances the sharpness and intensi-ies of the D and G bands due to the Raman enhancement. The metaleposition generally increases the surface area of the grapheneano structures. This is evidenced by the enhanced redox activity

or the [Fe(CN)6]3−/4− on the ITO/ErG/AuNP (lowest �Ep 75 mV andncreased ipa 51 �A, curve c) than the ITO (�Ep: 183 mV, ipa: 29 �A)nd ITO/ErG surfaces (�Ep: 275 mV, ipa: 22 �A, curve b), Fig. 6A.he extensive decrease of �Ep suggests that the AuNPs are wellnteracting with the functional groups, defects, ITO boundaries andnhances the conductivity of the ITO/ErG film. The co-deposition ofraphene and AuNP by the potential cycling method using the GOnd HAuCl4 (1 mg mL−1 and 0.5 mM in PBS buffer) solution resultedn the unstable film formation on the ITO surface. Similar observa-ion holds true for the ITO/ErG/PtNP films as well. This confirmshe fact that the sequential deposition of the graphene followed byhe AuNP deposition is the only suitable method to form a stableraphene–metal composite on the ITO plate.

Although antibodies could be directly immobilized on theuNPs, the non-covalent immobilization prone to induce the non-

pecific and improper orientation leading to data instability ofhe sensor, and the covalent immobilization using thiol linkages the most preferred. Hence, the MPA molecule is used as thenchoring layer for the antibody on the AuNP. For this, the MPA is

Acta 114 (2013) 590– 597 595

immobilized on the AuNP via the simple gold–thiol interaction[44,47–50]. The MPA enhances electrode blocking effect (the acidgroups exert high negative charges at pH 7.4 (pKa 5.2)) throughelectrostatic repulsion toward the [Fe(CN)6]3−/4−. The carboxylgroup activation by the EDC/NHS decreased the peak current(38 �A) and increased the �Ep (125 mV), Fig. 6B, curve d. Immo-bilization of anti-estradiol-antibody on the MPA layer throughpeptide coupling on the ITO/rGO/AuNP/MPA again decreased thepeak current (32 �A) and increased the �Ep (194 mV), Fig. 6B, curvee. The antibody interaction with the target antigen 17�-estradiol(1 �M) further reduces extensively the peak current (32 to 18 �A)and enhances the �Ep (307 mV) suggesting the effective formationof the antibody–antigen complex on the electrode surface, Fig. 6B,curve e. The regular increase of quasi reversible behavior of the[Fe(CN)6]3−/4− is attributed to the increased molecular crowdingand negative charge density at the electrode/film interface, Fig. 6Band Scheme 1. The control experiment conducted in parallel usingthe estrone (E1) and estriol (E3) as the target antigens on othersensor surfaces constructed under similar experimental conditionsshowed an insignificant signal change for all concentrations stud-ied indicating the high selectivity of the sensor developed. The CVbehaviors of the E2 and E3 are given in Fig. S8A and B, respec-tively. The reproducibility and renewability characteristics of thesensor are validated by de-hybridizing the antibody-antigen com-plex using 7 M urea. The CV profile of the de-hybridized surface isconstant and stable for six different concentration measurements.New surface is constructed and used for studying the new con-centration of the target antigen. The effect of varying the targetestradiol concentration on the ipa is presented in Fig. 7. The linearfit is made and the resulted regression equation is ipa = −0.5 × 10−4

[C] + 0.0021 with coefficient r2 0.89. The CV peak change with E2concentration is presented in Fig. S9. Further change in target con-centration does not have significant effect on the CV behavior.Storage of the sensor more than a week’s time deteriorates the per-formance to 55%, Fig. S10 and, hence, construction of new sensorsurface is essential.

The sensor showed a linear variation of ipa with target con-centrations ranging from 1 × 10−3 to 0.1 × 10−12 M with thelowest concentration detection 0.1 fM. Haque et al. [27] have

Fig. 7. Effect of estradiol antigens concentration on the CV peak current of 1 mM[Fe(CN)6]3−/4− in phosphate buffer at a scan rate 20 mV s−1 in the potential window−0.6 to 0.7 V (curve a). The control antigen Estrone behavior is shown as curve b infigure is measured using different bioelectrode prepared under similar experimentalconditions.

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etection limit 1.0 × 10−13 M using the gold transducer, in whichhe antibody is immobilized on the MPA layer on the gold electrodeor immunosensing. One order lower detection limit observed inhe present work may be attributed to thick graphene layer forma-ion which reduces the conductivity.

. Conclusions

The ErG/AuNP composite is prepared by the simple potentialycling method directly on the ITO surface for label free immunoensing of estradiol selectively and sensitively in phosphate buffer.he spectroscopic results indicate that the use of continuous poten-ial cycling method produces thick graphene with flat and verticalrientation on the ITO surface. The continuous potential cyclingethod reduces selectively the acid functionals of the GO at pH 6.5

indicated by XPS) and decreasing the charge transfer rate for bothFe(CN)6]3−/4− and [Ru(NH3)]2+/3+. Absence of acid functional groupimits the direct immobilization of anti-estradiol antibody. Hence,he deposition of AuNP is essential to immobilize biomolecules.he AuNP affects the ErG film morphology by inducing changef flat oriented ErG into vertical orientation and enhances elec-ronic property. The nature of AuNPs interaction on the ITO/ErG isecided by the size of tin oxide nano dots and the hetero junctionsormed between the tin oxide nano dots (seen in the FESEM, Fig. S3f the bare ITO and ErG/AuNP Fig. 1B) and graphene layers whenhe GO and Au3+ ions are reduced sequentially on the ITO. This isevealed by the RS, CV and FESEM techniques. Although, the sensorhowed the lowest detection limit 0.1 fmol, the elimination of largeackground signal to improve the sensitivity and data reliability isecessary. The results suggest that the continuous and sequentialotential cycling method is an alternative and simple approach forhe control of electronic property of ErG films on the ITO surfaceor sensor materials development at the reduced cost, however,omplete reduction of hydroxyl and epoxide groups is essential forchieving high sensitivity. Hence, further work is in progress on themprovement of the sensor by controlling the graphene layer thick-ess and orientation of both the graphene layer and antibody for

mproving the selectivity and sensitivities of sensors of these types.he antibody used in this study has high affinity for estradiol thanhe estrogen receptors which is very stable and readily available inufficient quantity than the estrogen receptor.

cknowledgements

Venkataraman Dharuman gratefully acknowledge the Indianouncil of Medical Research, DHR, New Delhi, India for theward of Fellowship for Long Term Research Training in For-ign Research Institute for the year 2012–2013 (ICMR Award no.EC/DHR/HRD-FELLOW/1(6)/2011, Dated 14.08.2012). J.H. Hahnreatly acknowledges the financial support from the Basic Scienceesearch Program (2012-0007102) of the National Research Foun-ation (NRF), the Ministry of Education, Science and Technology,outh Korea.

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/.electacta.2013.10.128.

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