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Sensors and Actuators B 188 (2013) 1201– 1211

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journa l h om epage: www.elsev ier .com/ locat e/snb

novel impedimetric biosensor based on graphene oxide/goldanoplatform for detection of DNA arrays

inod Kumar Guptaa,b,∗, Mehmet Lütfi Yolac,d, Munewar Saeed Qureshie,li Osman Solak f,g, Necip Atare, Zafer Üstündage

Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247667, IndiaDr. R M L Avadh University Faizabad, UP, 224001, IndiaSinop University, Faculty of Arts and Sciences, Department of Chemistry, Sinop, TurkeyHacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, Ankara, TurkeyDumlupinar University, Faculty of Arts and Sciences, Department of Chemistry, Kutahya, TurkeyAnkara University, Faculty of Science, Department of Chemistry, Ankara, TurkeyKyrgyzstan-Turkey Manas University, Faculty of Engineering, Department of Chemical Engineering, Bishkek, Kyrgyzstan

r t i c l e i n f o

rticle history:eceived 30 April 2013eceived in revised form 24 July 2013ccepted 7 August 2013vailable online xxx

eywords:raphene oxide

a b s t r a c t

A highly sensitive method for detection of DNA hybridization was developed. This method wasbased on the modification of glassy carbon electrode with gold nanoparticles (AuNPs) involving p-aminothiophenol (ATP) functionalized graphene oxide (GO). This GO was used as a platform forimpedimetric genosensing using 5′-TA GGG CCA CTT GGA CCT-(CH2)3-SH-3′ single-stranded probe (ss-DNA), 5′-AGG TCC AAG TGG CCC TA-3′ (target DNA), 5′-SH-C6-TAG GGC CA-3′ (non-complementary-1)and 5′-SH-C6-TGC CCG TTA CG 3-′ (non-complementary-2) oligonucleotide sequences. The film exhibitedexcellent properties for immobilizing DNA probes and sensing DNA hybridization. The DNA immobi-

old nanoparticleslectrochemical impedance spectroscopyNA nanobiosensor

lization and hybridization on the film were studied by cyclic voltammetry (CV), and electrochemicalimpedance spectroscopy (EIS), and found that the charge transfer resistance (Rct) of the electrodeincreased with the concentration of the target DNA hybridized with the ss-DNA. The linear detectionrange was from 1.0 × 10−13 M to 1.0 × 10−7 M and the detection limit was 1.10 × 10−14 M (n = 6). Comparedwith the other electrochemical DNA biosensors, the proposed biosensor showed its own performance ofsimplicity, good stability, and high sensitivity.

. Introduction

Since the inception of the concept of DNA biosensor [1], itsevelopment has attracted substantial attention in connection withesearch efforts directed at gene analysis, the detection of geneticisorders, tissue matching, forensic applications, etc. [2–7]. As com-ared to other techniques, electrochemical sensors have receivedonsiderable attention due to their fast response, remarkably highensitivity, good selectivity, and strong operability [8–13].

Graphene/graphene oxide has attracted strong scientific andechnological interest in recent years [14–19]. It has shown great

romise in many applications, such as electronics [20], energy stor-ge and conversion supercapacitors [21], batteries [22,23], fuelells [24–28], solar cells [29,30], and bioscience/biotechnologies

∗ Corresponding author at: Department of Chemistry, Indian Institute of Technol-gy Roorkee, Roorkee 247667, India. Tel.: +91 1332285801;ax: +91 1332273560 36.

E-mail addresses: vinodfcy@iitr.ernet.in, vinodfcy@gmail.com (V.K. Gupta).

925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.snb.2013.08.034

© 2013 Elsevier B.V. All rights reserved.

[31–36] because of its unique physicochemical properties, whichinclude high surface area (theoretically 2630 m2 g−1 for single-layergraphene) [14,18], excellent thermal conductivity [37], electricalconductivity [14,38], and strong mechanical strength [39].

Several electrochemical sensors based on graphene andgraphene composites for bioanalysis and environmen-tal analysis have been developed [33,40]. Shan et al. [33]reported the first graphene-based glucose biosensor with agraphene/polyethylenimine-functionalized ionic liquid nanocom-posite modified electrode that exhibited a linear glucose response(2–14 mM, R2 = 0.9940). Zhou et al. [40] reported a glucosebiosensor based on chemically reduced graphene oxide (CR-GO). CR-GO-based biosensor exhibits substantially enhancedamperometric signals for sensing glucose with wide linearrange (0.01–10 mM), high sensitivity (20.21 mA cm−2), and a lowdetection limit of 2.00 �M (S/N = 3).

To improve DNA hybridization efficiency, different kinds ofnanomaterials have been applied for electrode surface modifi-cation due to their increased surface area, and their beneficialorientation effect for DNA immobilization and recognition [41,42].

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mong all the nanomaterials, AuNPs are the most frequently usedor electrode surface modification in the fabrication of biosensors43,44]. Cai et al. [45] assembled the AuNPs (16 nm in diameter)n a cysteamine-modified gold electrode and discovered that themmobilization quantities of thiolated probe DNA on the modifiedlectrode were increased more than that on a bare gold electrode.iang’s group [46,47] also utilized a AuNPs modified electrode tonhance the immobilization of DNA and ability for hybridization.he other nanoparticles such as ZnO [48], FePt/ZnS nanocore–shell49], a mercaptoacetic acid-modified cadmium sulfide [50] andarbon-nanotubes/nano zirconium dioxide/chitosan [51] were pre-ared and used for providing DNA or nucleic acid hybridization.

Graphene/metal nanoparticle based biosensors have also beeneveloped. Shan et al. [52] reported a graphene/AuNPs/chitosanomposite film based biosensor that exhibited good electrocatalyt-cal activity toward H2O2 and O2. Wu et al. [53] reported a glucosexidase/graphene/PtNPs/chitosan biosensor with a detection limitf 0.6 mM glucose. This enhanced performance was attributed tohe large surface area, good electrical conductivity of graphene,ynergistic effect of graphene, and metal nanoparticles [52,53].

Liu designed the graphene oxide platform for oligonucleotideased DNA sensor in the presence of the various salt concentra-ions. He reported the sensor response in detail for the optimumarameters such as pH factor, salt concentration, hybridizationime, temperature, etc. [54]. The uncharged surfaces allowed the

olecules to hybridize to DNA strands with high affinity and speci-city [55]. To prevent the electrostatic repulsion, NaCl solution wasdded to the medium. Total charge amount was adjusted using NaCl54,56]. In this study, we preferred 0.05 M phosphate buffer (pH 7.4)ontaining 20 mM NaCl at room temperature. The experimentalonditions were in agreement with the literatures [54,56].

In the present paper, a highly sensitive method for detectionf DNA hybridization was developed based on the glassy car-on electrode modified with AuNPs involved on GO. The GO anduNPs were prepared and characterized by transmission elec-

ron microscope (TEM), X-ray photoelectron spectroscopy (XPS),V–vis spectroscopy, reflection-absorption infrared spectroscopy

RAIRS), atomic force microscopy (AFM) and the X-ray diffractionXRD) method. The performance of the developed biosensor wasptimized by determination of DNA under suitable analytical con-itions. In addition, this study indicates that graphene oxide/goldanoparticles nanomaterial has important role in electrochem-

cal sensors and biosensors. When we consider the advantagesf the special physical or chemical properties of this nanoma-erial, improved electrochemical sensors and biosensors can beonstructed.

. Material and methods

.1. Apparatus and reagents

All chemicals were reagent grade and used as receivednd included the following: HPLC grade acetonitrile (MeCN)Sigma–Aldrich), isopropyl alcohol (IPA) (Sigma–Aldrich),ctivated carbon (Sigma–Aldrich), potassium ferricyanideK3Fe(CN)6) (Sigma–Aldrich), potassium ferrocyanide (K4Fe(CN)6)Sigma–Aldrich), hydrogen tetrachloroaurate (HAuCl4)Sigma-Aldrich), p-aminothiophenol (ATP) (Sigma–Aldrich), N-(3-imethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDC)Sigma–Aldrich), trisodium citrate dehydrate (Na3C6H5O7·2H2O)Sigma–Aldrich), potassium chloride (KCl) (Merck), graphite

Merck), sulfuric acid (H2SO4) (Merck), hydrogen peroxide (H2O2)Merck), ethanol (Merck), potassium permanganate (KMnO4)Merck), potassium persulfate (K2S2O8) (Merck), and phosphorusentoxide (P2O5) (Merck).

rs B 188 (2013) 1201– 1211

All the processes were performed in aqueous media and thepreparation of the aqueous solutions were carried out using ultrapure quality water with a resistance of 18.3 M� cm (Human Power1+ Scholar purification system). The oligomers were obtained fromElla Biotech GmbH (Germany). Their base sequences were as fol-lows:

Single-stranded probe (ss-DNA): 5′-TA GGG CCA CTT GGA CCT-(CH2)3-SH-3′.Target DNA: 5′-AGG TCC AAG TGG CCC TA 3-′.Non-complementary-1 (NC1-DNA): 5′-SH-C6-TAG GGC CA-3′.Non-complementary-2 (NC2-DNA): 5′-SH-C6-TGC CCG TTA CG-3′.

DNA oligonucleotide stock solutions (100 �M DNA) were pre-pared in ultra pure quality water and were stored at −20 ◦C. Dilutesolutions of the oligonucleotides were prepared daily with 0.05 Mphosphate buffer containing 20 mM NaCl (pH 7.4).

The absorption spectra of AuNPs, the GO, and the modifiedGO were recorded with an Schimadzu UV2550 UV/Visible spec-trophotometer (Japan) having a photodiode array detector. HRTEMmeasurements were performed on a JEOL 2100 HRTEM instrument(JEOL Ltd., Tokyo, Japan).

Infrared spectra of the nanostructures were recorded at roomtemperature (Bruker Tensor 27 FT-IR) and a DTGS detector by usinga Ge total reflection accessory (GATR; 65◦ incident angle relative tosurface normal, Harrick Scientific).

XPS analysis was performed on a PHI 5000 Versa Probe (�ULVAC-PHI, Inc., Japan/USA) model X-ray photoelectron spectrom-eter instrument with monochromatized AlK� radiation (1486.6 eV)as an X-ray anode operated at 50 W. The pressure inside theanalyzer was maintained at 10−7 Pa. The samples for XPS measure-ments were prepared on a clean glass slide by placing one drop ofthe nanostructure and then allowing them to dry in air.

A Rikagu Miniflex X-ray diffractometer, using mono-chromaticCuK� radiation and operating at a voltage of 30 kV and a current of15 mA, was used for X-ray diffraction measurements of the nano-structures. A scanning speed of 2◦ 2�/min and a step size of 0.02◦

were used to examine the samples in the range of 5–75◦ 2�.Atomic force microscopy imaging was monitored by using a

AFM (Ntegra with Solaris plat-form, manufactured by NT-MDT)microscopy. Bare glassy carbon and modified glassy carbon surfacewere compared with the AFM 3D imaging.

Before electrochemical experiments, solutions were purgedwith pure argon gas (99.999%) for at least 10 min and an argonatmosphere was maintained over the solution during experiments.All electrochemical experiments were performed using a GamryReference 600 work-station (Gamry, USA) electrochemical ana-lyzer equipped with a C3 cell stand.

The working electrode was a bare or modified glassy carbonelectrode (GCE) (BAS, MF-20) with a geometric area of 0.027 cm2.The reference electrode was Ag/AgCl/KCl (sat) in aqueous mediaand the counter electrode was a Pt wire.

Electrochemical impedance spectroscopic experiments werecarried out with a Gamry Reference 600 work-station equippedwith a PCI4/300 potentiostat in conjunction with EIS 300 software.Modified electrodes were characterized in 1.0 mM [Fe(CN)6]3−/4−

(1:1) redox couple via EIS methods. EIS data was measured at300 kHz to 0.1 Hz at 10 mV wave amplitude and at an electrodepotential of 0.165 V, the formal potential of the 1 mM [Fe(CN)6]3−/4−

(1:1) redox couple. The reported results for every electrode in thispaper were the mean value of six parallel measurements.

2.2. Cleaning procedure for the glassy carbon electrode surface

Glassy carbon electrodes were cleaned and prepared by polish-ing them to a mirror-like finish with fine wet emery paper (grain

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V.K. Gupta et al. / Sensors and A

ize 4000). They were polished successively in 0.1 �m and 0.05 �mlumina slurries (Baikowski Int. Corp., USA) on microcloth padsBuehler, Lake Bluff, IL, USA). The electrodes were sonicated twicen ultra pure water and then in 50:50 (v/v) IPA and MeCN solu-ion, purified twice over activated carbon. After removal of tracelumina from the surface by rinsing with water and a brief clean-ng in an ultrasonic bath (Bandelin RK 100, Germany) with water,PA + MeCN mixture was then purified over the activated carbon,nd GCE was rinsed with MeCN to remove any physisorbed, unre-cted materials from the electrode surface.

.3. Preparation of AuNPs

HAuCl4 and Na3C6H5O7·2H2O were used as a gold precursornd a reducing agent, respectively. 20 mL of 1.0 mM HAuCl4 wasdded into a 50 mL volumetric flask on a stirring hot plate. Whiletirring, 2 mL of Na3C6H5O7·2H2O (1%) was slowly added into theold precursor solution at 65 ◦C. The solution was well mixed at theame temperature until the color of the solution changed to red;t was then boiled for 20 min. The mean diameters of the AuNPsre in the range of 8–10 nm and the AuNPs were stabilized withthanol [57].

.4. Preparation of GO

GO was synthesized by a modified Hummers method [58–60]. mixture containing 25 mL of H2SO4 (98%) with 5 g of K2S2O8, 5 gf P2O5, and 5 g of graphite was placed in a flask and was keptt 80 ◦C for 6 h. The mixture was cooled to 20 ◦C and diluted with

L of ultra pure water and left for 12 h. The pre-oxidized carbonaterial was filtered and washed with ultra pure water. The pre-

reated graphite was diluted with 250 mL of H2SO4 (98%) under◦C. 30 g of KMnO4 was added in the suspension and cooled to0 ◦C. After the KMnO4 feeding was finished, the flask was heated tobout 35 ◦C and kept at this temperature for an additional 30 min.he mixture was stirred under this temperature for 4 h and theniluted with 500 mL of ultra pure water in the ice bath. The lastixture was stirred for 2 h. The mixture was diluted to 2 L of ultra

ure water. The suspension was then further treated with 40 mLf H2O2 (30%). The color of the suspension changed from brownisho brilliant yellow and the mixture was stirred until the bubblingtopped. The synthesized GO was then filtered and washed with

Scheme 1. The EIS measurement diagram of ss-DNA/A

rs B 188 (2013) 1201– 1211 1203

0.1 M HCl and ultra pure water three times, respectively. The GOwas precipitated by using an ultracentrifuge and was dried underthe atmospheric air conditions.

2.5. Fabrication of AuNPs-ATPGO/GCE

The synthesized GO was dissolved in ethanol at a concentra-tion of 2 mg mL−1 with the aid of ultrasonic agitation for 1 h,resulting in a homogeneous black suspension. To ensure surfaceactivation of carboxylate groups of GO, the GO suspension wasinteracted with 0.2 M EDC solution for 8 h. Activated GO suspen-sion was mixed well with 1.0 mM ATP at a 1:1 volume ratio for2 h (ATPGO). In a typical experiment of self-assembly, the aqueousdispersion of AuNPs (1 mg mL−1) was mixed with the aqueous dis-persion of ATPGO sheets (0.1 mg mL−1) at a 1:1 volume ratio andsonicated for 15 min to form a homogeneous mixture. The mix-ture was then kept undisturbed under ambient conditions for 12 h(AuNPs-ATPGO). Prior to the surface modification, GCE was pol-ished successively in 0.1 �m and 0.05 �m alumina slurries, thenultrasonically cleaned in ultra pure water twice in 50:50 (v/v)IPA, and finally purified twice with MeCN solution over activatedcarbon. The AuNPs-ATPGO/GCE was prepared by casting a 10 �Laliquot of 2 mg mL−1 AuNPs-ATPGO suspension onto the surface ofcleaned GCE and then evaporating the solvent under an infraredlamp.

2.6. Immobilization of ss-DNA probes on the AuNPs-ATPGO/GCEand hybridization

The ss-DNA probes could be immobilized on the AuNPs-ATPGO/GCE surface by immersing the electrode in 1.0 × 10−6 Mss-DNA probe solution for 60 min. The ss-DNA probe was immo-bilized onto the surface of AuNPs-ATPGO/GCE by the affinity ofAuNPs for biomolecules. After the electrode was washed withwater to remove the non-immobilized ss-DNA, the captured probeelectrode (ss-DNA/AuNPs-ATPGO/GCE) was ready for use. The ss-DNA/AuNPs-ATPGO/GCE was transferred into the hybridizationsolution containing different concentrations of target DNA for

90 min at 35 ◦C. After that, the electrode was rinsed three times withwater to remove the non-hybridized target. Scheme 1 illustrates theschematic diagram of the structure of ss-DNA/AuNPs-ATPGO/GCEhybridized with target DNA.

uNPs-ATPGO/GCE hybridized with target DNA.

1204 V.K. Gupta et al. / Sensors and Actuators B 188 (2013) 1201– 1211

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ig. 1. (A) TEM image of the GO. (B) TEM image of the AuNPs-ATPGO. (C) UV–vis snd (c) ATPGO.

. Results and discussion

.1. Characterization of GO, ATPGO and AuNPs-ATPGOomposites

GO and the AuNPs-ATPGO composites were first characterizedorphologically using JEOL 2100 HRTEM with an accelerating volt-

ge of 200 keV. Samples were deposited on a polymeric grid andried at room temperature under an argon gas stream. The trans-arent and wrinkled GO sheets in Fig. 1A have exhibited mono- orew-layer planar sheet-like and pellucid morphology. AuNPs in theanocomposite has been seen as dark dots with a mean diameterf 12–20 nm on a lighter shaded substrate corresponding to thelanar GO sheet (Fig. 1B).

The AuNPs-ATPGO formation was confirmed by UV–vis spec-roscopy (Fig. 1C). The UV–vis spectrum of the GO in ethanol showshat no absorption peak exists in the range of 450–800 nm (curve

of Fig. 1C). As shown in curve b of Fig. 1C, the successful syn-hesis of AuNPs is confirmed by the presence of a peak at 522 nm,hich is typical for AuNPs [61]. When the AuNPs were attached

o the ATPGO, the absorption peak of AuNPs in the AuNPs-ATPGOomposite shifted to 520 nm and a small change in shape and inten-ity occurred (curve c). The absorption bands in this region arettributed to electronic transitions in the organic moieties attachedn the nanoparticles.

IR measurements were carried out to show the formation of theO and ATPGO. No IR peak exists in the range of 4000–600 nm in thepectrum of the graphite (curve a of Fig. 1D). In the spectrum of theO (curve b) in Fig. 1D, the bands around 3290 and 1739 cm−1 arettributed to the oxygen-containing functional groups on the GO

62], indicating the successful oxidation of the graphite. The IR spec-ra of GO shows the stretching vibrations of C O (1739 cm−1) on thedges of the GO planes [63]. The IR spectra (curve c) of the ATPGOhown in Fig. 1D confirm the presence of ATP covalently bonded

of (a) GO; (b) AuNPs; (c) AuNPs-ATPGO. (D) IR spectra of the (a) graphite; (b) GO

to GO. The peak at 1739 cm−1 corresponding to the vibrational fre-quency of C O in GO shifted to 1747 cm−1 in ATPGO. This shift isattributed to covalent functionalization of the carboxyl group of theGO with ATP.

Attachment of AuNPs on the ATPGO surface to construct AuNPs-ATPGO composite was identified by XPS experiments. In the XPSspectrum of the AuNPs-ATPGO composite (Fig. 2A), C, N, S, andAu peaks are observed. After the chemical attachment of theseorganic moieties, the peak at 285.6 eV, which is due to the sp2

hybridized C atoms [64], is transformed into a broad envelope anddeconvolution reveals that it consists of two components for theAuNPs-ATPGO composite. When the Cls region is curve-fitted, a freeCOOH group of the GO is located at 286.7 eV. The peaks at 285.6 and283.7 eV are assigned to C O/C N and C H carbon bonding ener-gies, respectively [65–67]. The XPS N1s narrow region spectra ofthe AuNPs-ATPGO composite is curve-fitted and the peak locatedat 399.6 eV is attributed to the presence of amide’s N H groups inthe covalent functionalization of the carboxyl group of the GO withthe amino group of the ATP [65].

S2p region is characterized by a doublet (2p1/2 and 2p3/2), owingto the spin-orbit coupling. It is thought that the sulphur of theAuNPs-ATPGO composite was easily bonded to the AuNPs by theappearance of sulphur peaks at 167.5 eV. The peak at 163.0 eV indi-cated a free mercapto group [68]. The Au 4f7/2 peak signal appearedat 83.10 eV and verified the presence of bonded Au [69].

The synthesis of the AuNPs-ATPGO composite was alsoevidenced by XRD measurements. The XRD patterns of the graphiteand as-synthesized AuNPs-ATPGO composite are examined andshown in Fig. 2B. As shown in the inset of Fig. 2B, the XRD pat-terns of the graphite and as-synthesized AuNPs-ATPGO reveal a

very intense and narrow peak at 2� = 26.5◦, assigning to the (0 0 2)planes of GO layers occurring in graphite. Three weak peaks at2� = 42.5◦, 44.6◦, and 54◦ correspond to the (1 0 0), (1 0 1), and (0 0 4)planes, respectively, of GO layers occurring in graphite (inset of

V.K. Gupta et al. / Sensors and Actuators B 188 (2013) 1201– 1211 1205

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ig. 2. (A) The narrow region XPS spectra of AuNPs-ATPGO composite for the decomposite. Inset: XRD patterns of the graphite. (C) The atomic force microscopic imaurface.

ig. 2B) [70]. As shown in Fig. 2B, a broad peak at 2� = 20–28◦ occurs,hich occurs due to the structure expansion as oxygen-containing

roups incorporate between the carbon sheets during the course

f strong oxidation. Two peaks occurred at 2� = 23.0◦ and 42.8◦

hich is a typical XRD pattern of the GO. The peaks at 2� = 38.2◦

nd 44.1◦ indicate the (1 1 1) and (2 0 0) crystalline planes of Au71].

ution spectra of the C1s, N1s, S2p and Au4f. (B) XRD patterns of the AuNPs-ATPGOare glassy carbon surface. (D) The atomic force microscopic image of AuNPs-ATPGO

The AuNPs-ATPGO modified GCE and bare GCE were character-ized by using an AFM microprobe. In Fig. 2C and D, the modificationof AuNPs-ATPGO nanocomposite on GCE resulted in the forma-

tion of relatively rough surface prepared by self ordered under aninfrared lamp. The surface morphology of the bare GCE (Fig. 2C) wasmonitored to be of less roughness than the AuNPs-ATPGO modifiedGCE (Fig. 2D).

1206 V.K. Gupta et al. / Sensors and Actuators B 188 (2013) 1201– 1211

Fig. 3. (A) Cyclic voltammograms of 1.0 mM [Fe(CN)6]3−/4− (1:1) containing 0.1 M KCl at different electrodes with the scan rate of 200 mV/s. (a) bare GCE interacted with onlyAuNPs; (b) the ss-DNA/AuNPs-ATPGO/GCE; (c) the ss-DNA/AuNPs-ATPGO/GCE hybridizing with 1.0 × 10−9 M target DNA. (B) Nyquist plots recorded at different electrodes( ted ws .0 mM

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a) bare GCE interacted with only AuNPs; (b) the ss-DNA/AuNPs-ATPGO/GCE interacs-DNA/AuNPs-ATPGO/GCE hybridizing with 1.0 × 10−13 M target DNA. Medium is 1

.2. CV and EIS of DNA hybridization ofs-DNA/AuNPs-ATPGO/GCE

In order to monitor the results of the different modified elec-rodes, the cyclic voltammograms of 1.0 mM [Fe(CN)6]3−/4− (1:1)t different modified electrodes were investigated (Fig. 3A). Itould been seen that 1.0 mM [Fe(CN)6]3−/4− (1:1) had a reversibleoltammogram with redox peaks in the potential range of 0.0/0.6 VFig. 3A, curve a). The bare GCE interacted with only AuNPs.owever, the reversible redox peaks were almost suppressedn ss-DNA/AuNPs-ATPGO/GCE interacted with NC1-DNA and ss-NA/AuNPs-ATPGO/GCE hybridized with target DNA (curve b andurve c of Fig. 3A).

EIS is an effective method for probing the features of modi-ed surfaces. Fig. 3B shows the impedance plots of the surfaces

n the form of nyquist diagrams. The nyquist diagrams at the ss-NA/AuNPs-ATPGO/GCE interacting with NC1-DNA and NC2-DNA

how an almost straight line of approximate unit slope with amall semicircle at the low frequency region (curve b and curve

of Fig. 3B). Thereby, indicating that diffusion is of a limitedtep with a low charge transfer resistance. The nyquist diagramfter the hybridization (curve d of Fig. 3B) shows only a semi-ircle with an approximate diameter of 23 k�. Higher chargeransfer resistance after the hybridization as compared to that oft the ss-DNA/AuNPs-ATPGO/GCE surface is consistent with the

yclic voltammetric results. So we can say that the ss-DNA/AuNPs-TPGO/GCE hybridized with target DNA can decelerate electron

ransfer for [Fe(CN)6]3−/4− redox probe as compared with bare GCEnd the ss-DNA/AuNPs-ATPGO/GCE interacted with NC-DNA.

ith NC1-DNA; (c) the ss-DNA/AuNPs-ATPGO/GCE interacted with NC2-DNA; (d) the [Fe(CN)6]3−/4− (1:1) in 0.1 M KCl.

Fig. 4A shows the electron transfer reaction of ferro/ferricyanide(Fe(CN)6

3−/4−) in 0.1 M KCl on the ss-DNA/AuNPs/ATPGO/GCEhybridized with 1.0 × 10−13 M target DNA which is harmoniouswith the standard Randles equivalent to model circuit in thehigh frequency range. Equivalent circuit comprises the solutionresistance (Rs), the charge transfer resistance (Rct), the War-burg resistance (Wd) and the constant phase element (CPE) forthe ss-DNA/AuNPs-ATPGO/GCE hybridized with 1.0 × 10−13 M tar-get DNA. The experimental impedance values are matched withRandles equivalent circuit with a Warburg element simulationusing Gamry software (EIS 300 Electrochemical Impedance Spec-troscopy Software). R0

ct and Rct values were calculated fromthe impedance plots for Fe(CN)6

3−/4− at bare GCE and the ss-DNA/AuNPs-ATPGO/GCE hybridized with 1.0 × 10−13 M target DNAas 0.3 k� and 23 k�, respectively. The charge transfer resistanceat the ss-DNA/AuNPs-ATPGO/GCE hybridized is higher than thatat the bare GCE as estimated, indicating the passivation propertyof the film. Another important point is that high Rct value on themodified electrode shows the blocking effect of the ss-DNA/AuNPs-ATPGO/GCE hybridized and the electron transfer reaction forFe(CN)6

3−/4− is irreversible.

3.3. Optimization of experimental conditions

3.3.1. Effect of the GO concentration

The concentration of GO assembled on the electrode sur-

face influences the EIS response after ss-DNA/AuNPs-ATPGO/GCEhybridized with 1.0 × 10−9 M target DNA. The results showed thatwhen the concentration of GO exceeded 2 mg mL−1, Rct values of

V.K. Gupta et al. / Sensors and Actuators B 188 (2013) 1201– 1211 1207

F t the ss-DNA/AuNPs-ATPGO/GCE hybridizing with 1.0 × 10−13 M target DNA: Inset is theR olid one is nyquist plot for the randles equivalent circuit. Frequency range is from 300 kHzt e effect of hybridization time. Medium is 1.0 mM [Fe(CN)6]3−/4− (1:1) in 0.1 M KCl.

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Table 1Data of the calibration curve for the proposed method (n = 6).

Regression equation y = 28.03x + 388.90

Standard error of slope 0.24Standard error of intercept 0.16Correlation coefficient (r) 0.9992Linearity range (M) 1.0 × 10−13 to 1.0 × 10−7

Number of data points 7

ig. 4. (A) Fitting of impedance data for 1.0 mM [Fe(CN)6]3−/4− (1:1) in 0.1 M KCl aandles equivalent circuit. Dotted curve is the experimental nyquist plot while the so 0.1 Hz. (B) (a) the effect of GO concentration; (b) the effect of temperature; (c) th

he electrode stayed almost constant. So 2 mg mL−1 was recom-ended as a suitable concentration of GO in further experiments

Fig. 4B, curve a).

.3.2. Effect of the temperatureThe results revealed that the biosensor performance was con-

iderably affected by the temperature change. Rct values of thelectrode hybridized with 1.0 × 10−9 M target DNA were investi-ated using the temperatures of 10–45 ◦C (Fig. 4B, curve b). It wasound that Rct values were the highest at 35 ◦C; thereby achieving

steady-state when the temperature exceeded 35 ◦C. It is likelyhat the temperature increased the movement of the moleculesnd kinetic energy to transport them on the electrode surfacesffectively. So the optimized temperature was chosen as 35 ◦C.

.3.3. Effect of the hybridization time

The time for hybridization was also investigated in the range

f 30–120 min. The results showed that at the concentration of.0 × 10−9 M target DNA, the highest Rct value was obtained whenhe time for hybridization was 90 min. For a longer time, Rct value

y = ax + b, y: �Rct (k�), x: target DNA oligonucleotide concentration (log c, mol/L), a:slope, b: intercept.

did not increase and stayed almost constant, indicating that thess-DNA probes on the modified electrode had been hybridized bytarget DNA completely. So the hybridization was carried out for atleast 90 min (Fig. 4B, curve c).

3.4. Sensitivity of the ss-DNA/AuNPs-ATPGO/GCE surface

The sensitivity of the impedimetric biosensor was further inves-tigated by varying the target DNA oligonucleotide concentration by

1208 V.K. Gupta et al. / Sensors and Actuators B 188 (2013) 1201– 1211

Table 2Comparison of the performance of the proposed biosensor with other electrochemical DNA biosensor.

Composition of the electrodes Detection method Linear range (M) Detection limit (M) References

Au/CdNPs DPV 2.0 × 10−10 to 1.0 × 10−8 2.0 × 10−11 [12]Graphene/PANIw DPV 2 1.2 × 10−5 to 2.12 × 10−12 3.25 × 10−13 [72]MNP-Cys/CNTs-GNP/Chi EIS 1.0 × 10−9 to 1.0 × 10−6 1.0 × 10−9 [73]Ga2Se3-3MPA EIS 1.4 × 10−8 to 2.0 × 10−8 6.6 × 10−10 [74]AuNPs/PEM DPV 1.0 × 10−5 to 1.0 × 10−11 1.0 × 10−11 [75]PAN-nanoZrO2/PTyr EIS 1.0 × 10−13 to 1.0 × 10−6 2.68 × 10−14 [76]MWCNTs/Ptnano DPV 2.25 × 1 −11 −7 −11

MWCNTs/COOH DPV 2.0 × 10AuNPs-ATPGO EIS 1.0 × 10

Fig. 5. (A) EIS response of the ss-DNA/AuNPs-ATPGO/GCE after hybridization withincreasing concentration of target DNA. (B) The plot of �Rct vs. the logarithm ofthe target DNA concentration. �Rct was the difference of Rct recorded at the ss-DNA/AuNPs-ATPGO/GCE after and before the ss-DNA probe hybridized with targetD

E0ptai

cnti

[5] W.M. Zheng, L. He, Label-free real-time multiplexed DNA detection using flu-orescent conjugated polymers, Journal of the American Chemical Society 131

NA.

IS with the use of the redox probe 1.0 mM Fe(CN)63−/4− solution in

.1 M KCl. Fig. 5A shows that the Rct values obtained at an electrodeotential of 0.165 V were in direct proportion to the amount of thearget oligonucleotides varied from 1.0 × 10−13 M to 1.0 × 10−7 Mnd the detection limit was 1.10 × 10−14 M (n = 6). Data for the cal-bration curve (Fig. 5B) for the proposed method is given in Table 1.

The performance of the fabricated DNA biosensor has also beenompared with those reported in the literatures that have used

anostructured materials for the DNA immobilization layer andhe results are shown in Table 2. Compared with some specialnstrumental methods like PCR, it is still less sensitive, while the

0 – 2.25 × 10 1.0 × 10 [77]−10 to 5.0 × 10−8 1.0 × 10−10 [78]−13 to 1.0 × 10−9 1.13 × 10−14 This study

electrochemical detection is relatively simple, of low cost, and hassignificant sensitivity [79–97].

3.5. Stability and reproducibility of the DNA biosensor

To determine the reproducibility of the DNA biosensor, six dif-ferent modified electrodes were fabricated in the same procedure.The values of �Rct were recorded before and after the ss-DNAhybridized with 1.0 × 10−13 M target DNA. The relative standarddeviation of these six �Rct was 0.86%.

To understand the stability of the DNA biosensor, the modifiedelectrodes were incubated in a phosphate buffer of pH 7.4 at 35 ◦Cfor 72 h, followed by rinsing the electrode with ultrapure water.This was followed by hybridization with 1.0 × 10−13 M target DNAand measurement by EIS. The results showed that the incubatedelectrode had the same behavior as a new fabricated sensor. TheDNA hybridization sensor had good stability for 72 h.

4. Conclusions

We have developed a highly sensitive DNA electrochemicalbiosensor by modifying the GCE with AuNPs-ATPGO composite.CV and EIS techniques were successively employed to elucidatethe charge transfer changes occurring during the preparation andutilization of the developed DNA biosensor. Compared with otherelectrochemical DNA biosensors, the proposed biosensor has manyadvantages, such as simplicity, reproducibility, stability, and highsensitivity. Finally, the developed DNA biosensor can be used foranalysis of a series of real samples. Future studies in our laboratorywill be directed toward this aim.

Acknowledgment

The authors thank “Research Fellowship Programme for ForeignCitizens” of the “Scientific and Technological Research Council ofTurkey” (TUBITAK) for financial support.

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79] V.K. Gupta, A.K. Jain, G. Maheshwari, H. Lang, Copper (II)-selective potentio-metric sensor based on Porphyrins in PVC matrix, Sensors and Actuators B 117(2006) 99–106.

80] V.K. Gupta, A.K. Singh, S. Mehtab, B. Gupta, A Cobalt (II) selective PVC membranebased on a Schiff base complex of N, N′-bis (salicylidene)-3, 4-diaminotoluene,Analytica Chimica Acta 566 (2006) 5–10.

81] V.K. Gupta, A.K. Jain, P. Kumar, PVC-based membranes of N, N′-dibenzyl-1,4,10, 13-tetraoxa-7, 16-diazacyclooctadecane as Pb (II)-selective sensor, Sensorsand Actuators B 120 (2006) 259–265.

82] V.K. Gupta, A.K. Jain, P. Kumar, S. Agarwal, G. Maheshwari, Chromium (III)-selective sensor based on tri-o-thymotide in PVC matrix, Sensors and ActuatorsB 113 (2006) 182–186.

83] A.K. Jain, V.K. Gupta, S. Radi, L.P. Singh, J.R. Raisoni, A comparative study ofPb2+ sensors based on derivatized tetrapyrazole and calix[4]arene receptors,Electrochimica Acta 51 (2006) 2547–2553.

84] R.N. Goyal, V.K. Gupta, S. Chatterjee, A sensitive voltammetric sensor fordetermination of synthetic corticosteroid triamcinolone, abused for doping,Biosensors and Bioelectronics 24 (2009) 3562–3568.

85] V.K. Gupta, R.N. Goyal, R.A. Sharma, Comparative studies on Neodymium (III)-selective membrane sensors, Analytica Chimica Acta 647 (2009) 66–71.

86] R.N. Goyal, V.K. Gupta, S. Chatterjee, Fullerene – C60 – modified edge planepyrolytic graphite electrode for the determination of dexamethasone inpharmaceutical formulations and human biological fluids, Biosensors and Bio-electronics 24 (2009) 1649–1654.

87] R.N. Goyal, M. Oyama, V.K. Gupta, S.P. Singh, S. Chatterjee, Sensors for 5-hydroxytryptamine and 5-hydroxyindole acetic acid based on nanomaterialmodified electrodes, Sensors and Actuators B Chemical 134 (2008) 816–821.

88] R.N. Goyal, V.K. Gupta, N. Bachheti, R.A. Sharma, Electrochemical Sensor forthe Determination of Dopamine in Presence of High Concentration of AscorbicAcid using a Fullerene-C60 Coated Gold Electrode, Electroanalysis 20 (2008)757–764.

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92] R.N. Goyal, V.K. Gupta, A. Sangal, N. Bachheti, Voltammetric determination ofuric acid at a fullerene -C60-modified glassy carbon electrode, Electroanalysis17 (2005) 2217–2223.

93] V.K. Gupta, S. Chandra, H. Lang, A highly selective mercury electrode based ona diamine donor ligand, Talanta 66 (2005) 575–580.

94] V.K. Gupta, R. Prasad, A. Kumar, Preparation of ethambutol-copper (II) com-plex and fabrication of PVC based membrane potentiometric sensor for copper,Talanta 60 (2003) 149–160.

95] V.K. Gupta, S. Chandra, R. Mangla, Dicyclohexano-18-crown-6 as active mate-rial in PVC matrix membrane for the fabrication of cadmium selectivepotentiometric sensor, Electrochimica Acta 47 (2002) 1579–1586.

96] V.K. Gupta, R. Mangla, U. Khurana, P. Kumar, Determination of uranyl Ionsusing Poly (Vinyl Chloride) based 4-tert-butylcalix [6] arene membrane sensor,Electroanalysis 11 (1999) 573–576.

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Biographies

Vinod Kumar Gupta obtained his PhD degree in chem-istry from the University of Roorkee (now Indian Instituteof Technology Roorkee) Roorkee, India, in 1979. Sincethen he is pursuing research at the same Institute andpresently holding the position of Vice Chancellor, Dr. R ML Avadh University Faizabad, UP 224001, India. He workedas a post-doctoral fellow at University of Regensburg,Germany, in 1993 as an EC fellow and was DAAD visitingprofessor at University of Chemnitz and Freie University ofBerlin in 2002. He has published more than 350 researchpapers, many reviews and two books which fetched himmore than 17,500 citation with h-index of 88. He wasawarded the Indian Citation Laureate Award in 2004. His

research interests include chemical sensors, waste water treatment, environmentaland electroanalytical chemistry. Dr. Gupta is an elected Fellow of the World Inno-vation Foundation (FIWF) since July 2004 and Fellow of the National Academy ofSciences (FNASc) since 2008.

Mehmet Lütfi Yola is currently a research assistant andPhD student in Faculty of Pharmacy, Hacettepe Uni-versity, Ankara, Turkey. He received his MSc degree in2009 at Hacettepe University, Department of AnalyticalChemistry. His interests include Surface modification andcharacterization, Nanobiosensors, Biosensor applications,Biomaterials and Electroanalytical techniques.

Munawar Saeed Qureshi has completed PhD in Ana-lytical Chemistry from University of Sindh. Jamshoro,Pakistan in 2011. In 2009, He got six months Pre-DoctoralScholarship for Department of Analytical Chemistry atCharles University in Czech Republic through Interna-tional Research Support Initiative Program (IRSIP) fromHigher Education of Pakistan. Currently, He is working asPost-Doctoral fellowship by (TUBITAK) in Department ofAnalytical Chemistry, Dumlupinar University at Kutahya,His research interest areas are Micro and nano electrodespreparation, characterization and application for electro-analytical investigations of organic and inorganic species,Nanoparticles, Nanobiosensor.

Ali Osman Solak, Professor, is currently a senior lecturerat the Ankara University, Chemistry Department (Turkey).

visiting professor for two years between 2001 and 2003.

ctuato

Dumlupınar University, Chemistry Department. (Turkey).His research interests Surface Science, Nanoconstruc-tions, Nanobiosensors, Moletronics, Core@shell bimetallic

V.K. Gupta et al. / Sensors and A

Necip Atar received his MSc and PhD degrees in PhysicalChemistry from Dumlupinar University, Kutahya, Turkey.He is working at Dumlupinar University, Department ofChemistry, Kutahya, Turkey. He worked at Curtin Univer-

sity of Technology, Department of Chemical Engineering,Perth, Australia, as a visiting associate for one years;2009–2010. His research interests are surface science,cataylsis, nanoconstructions, nanobiosensors, nanoparti-cles and adsorption.

rs B 188 (2013) 1201– 1211 1211

Zafer Üstündag, PhD, is currently a senior lecturer at the

nanoparticles and Quantum Dots, SPM Imaging and Pat-terning Technology.