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Analytica Chimica Acta 767 (2013) 50– 58

Contents lists available at SciVerse ScienceDirect

Analytica Chimica Acta

jo u rn al hom epa ge: www.elsev ier .com/ locate /aca

sandwich-type DNA biosensor based on electrochemicalo-reduction synthesis of graphene-three dimensionalanostructure gold nanocomposite films

i-Lin Liua,e,1, Guang-Xian Zhonga,b,1, Jin-Yuan Chena,e, Shao-Huang Wenga,e,ong-Nan Huangd, Wei Chena,e, Li-Qing Lina,e, Yun Leia,e, Fei-Huan Fuf,hou-liang Sunc, Xin-Hua Lina,e,∗, Jian-Hua Linb,∗, Shu-Yu Yangc,∗

Department of Pharmaceutical Analysis, Faculty of Pharmacy, Fujian Medical University, Fuzhou 350004, PR ChinaDepartment of Orthopaedics, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350004, PR ChinaThe First Affiliated Hospital of Xiamen University, Xiamen 361003, PR ChinaDepartment of Physical and Chemical Analysis, Fujian Center for Disease Control and Prevention, Fuzhou 350001, PR ChinaNano Medical Technology Research Institute, Fujian Medical University, Fuzhou 350004, PR ChinaDepartment of Endocrinology, The County Hospital of Anxi, Anxi 362400, PR China

i g h l i g h t s

Graphene-three dimensional nano-structure gold nanocomposite modi-fied GCE is fabricated.A “sandwich-type” detection strat-egy is employed in this electrochem-ical DNA biosensor.The detection limit of the DNAbiosensor is 3.4 fM.The new DNA biosensor exhibiteda fast response, high sensitivity andselectivity.The new DNA biosensor has beenused for an assay of PCR real sample.

g r a p h i c a l a b s t r a c t

Graphite oxide (GO) Au-nanoparticle graphene capture probe biotinylated target probe avidin-HRPreporter probe.

r t i c l e i n f o

rticle history:eceived 19 October 2012eceived in revised form7 December 2012ccepted 30 December 2012vailable online 8 January 2013

a b s t r a c t

A novel electrochemical DNA biosensor based on graphene-three dimensional nanostructure goldnanocomposite modified glassy carbon electrode (G-3D Au/GCE) was fabricated for detection of sur-vivin gene which was correlated with osteosarcoma. The G-3D Au film was prepared with one-stepelectrochemical coreduction with graphite oxide and HAuCl4 at cathodic potentials. The active surfacearea of G-3D Au/GCE was 2.629 cm2, which was about 3.8 times compared to that of a Au-coated GCEunder the same experimental conditions, and 8.8 times compared to a planar gold electrode with a

eywords:raphene–gold nanostructureanocompositesNA biosensors

similar geometric area. The resultant nanocomposites with high conductivity, electrocatalysis and bio-compatibility were characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) andelectrochemical impedance spectroscopy (EIS). A “sandwich-type” detection strategy was employed inthis electrochemical DNA biosensor and the response of this DNA biosensor was measured by CV and

andwich-typeurvivin genesteosarcoma

amperometric current–time curve detection. Under optimum conditions, there was a good linear rela-tionship between the current signal and the logarithmic function of complementary DNA concentration

∗ Corresponding authors. Tel.: +86 591 22862016; fax: +86 591 22862016.E-mail addresses: xhl1963@sina.com (X.-H. Lin), jianhual@126.com (J.-H. Lin), yang.shuyu@yahoo.com.cn (S.-Y. Yang).

1 These authors contributed equally to the present study.

003-2670/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.aca.2012.12.049

A.-L. Liu et al. / Analytica Chimica Acta 767 (2013) 50– 58 51

in a range of 50–5000 fM with a detection limit of 3.4 fM. This new biosensor exhibited a fast amperometricresponse, high sensitivity and selectivity and has been used in a polymerase chain reaction assay of real-lifesample with a satisfactory result.

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. Introduction

In recent years, detection of particular gene mutations, copyumber variation, and single-nucleotide polymorphism haveecome extremely important for diagnosis and treatment of theene-related diseases. Electrochemical DNA biosensor, as a mon-toring technique, has been widely considered to be a promising

eans for diagnosis of genetic diseases and other biological anal-sis due to the rapid and sensitive response as well as theimple and convenient operation [1,2]. To develop a DNA biosen-or with high sensitivity and selectivity, nanomaterials such asanoparticles, nanoporous materials and nano-carbon materialsave been introduced as transducers for sensing enhancement3–5].

Graphene is a single-atom thick, two-dimensional sheet of sp2

onded carbon atoms, which are densely packed in a honeycombrystal lattice. Due to its good thermal conductivity, electricalonductivity, mechanical strength and extreme high surface area,raphene nanosheets hold great promise for potential applica-ions in various fields such as nanoelectronics [6,7], sensors [8–10],anocomposites [11,12], batteries [13,14], supercapacitors [15],nd hydrogen storage[16]. However, the practical applications ofraphene are challenged by its irreversible agglomeration bothn the drying state and in common solvents via � stacking [17].o further explore the application potential of graphene-basedaterials, various modification strategies have been proposed,

uch as decoration of graphene surface with metal nanoparticles,orming graphene–metal nanocomposites [18–20]. The preparedraphene–metal nanocomposites exhibit excellent electrocatalyticctivity and electrochemical stability for sensors and catalysts9]. The graphene–metal nanocomposites are usually prepared byhemical or thermal reduction of mixtures of graphene (or graphitexide; GO) and metallic precursors [18,21–23]. However, in theseethods, highly toxic reducing agents such as hydrazine hydrate

r hydroquinone are used, thus extreme care should be made. Inddition, the chemical synthesis processes are time-consuming andigh temperatures (95–100 ◦C) are required [24]. Recently, electro-hemical reduction of GO has attracted increasing attention dueo its simple, fast and green nature [25–27]. It has been reportedhat exfoliated GO precursor can be electrochemically reducedt cathodic potentials (completely reduced potential: −1.5 V vsg/AgCl) to form an electrochemically reduced GO (ERGO) [26]. Atuch negative potentials, metallic precursors such as HAuCl4 canlso be electrochemically reduced to form three-dimensional (3D)old nanostructures on a planar electrode [28]. Thus, it is possible torepare graphene–Au nanocomposite by a one-step electrochem-

cal coreduction at cathodic potentials. For example, Zhou et al.roposed a one-step electrochemical approach to the synthesis ofighly dispersed Pt nanoparticles (NPS) on graphene. The resultantraphene–Pt NPs nanocomposite showed higher electrocatalyticctivity and long-term stability toward methanol electrooxida-ion than Pt NPs/Vulcan [29]. Fu et al. prepared graphene–Auanocomposites in ionic liquid by electrochemical co-reductiont a constant potential of −0.2 V. The formed nanocompositesxhibited enhanced electrochemical activity and stability toward-hydroxytyramine hydrochloride (DA) [30]. Similarly, a one-stepethod for the synthesis of graphene–metal nanocomposite films

sing direct electrodeposition in a potential range from 0.6 to1.5 V is reported. In this method, graphene layers are regularly

paced by layers of Au nanoparticles, as a result, the conductivity

© 2013 Elsevier B.V. All rights reserved.

and surface area of the graphene–Au composite film are signifi-cantly improved compared to pure graphene film [17].

Osteosarcoma is the most common malignant bone tumor andit occurs most frequently during adolescence. Introduction of adju-vant and neoadjuvant results in better control of both local growthand metastatic spread of tumor cell [31,32]. However, resistance tochemotherapy associated with a high risk of relapse and a unde-sired outcome remains a major obstacle to successful treatmentof osteosarcoma [33,34]. Survivin is a member of the inhibitor ofapoptosis protein (IAP) family and is characterized by the presenceof single baculoviral IAP protein repeat (BIR) and lacks carboxylterminal RING finger domain in its protein structure [35,36]. Itis expressed highly in fetal tissue and most human tumors suchas gastric cancer, breast cancer, lung cancer, colorectal cancer,pancreatic cancer, urinary bladder cancer, soft tissue cancer, neu-roblastoma and osteosarcoma, but is completely absent in normaldifferentiated cells [37]. The survivin protein functions to inhibitcaspase activation, thereby leading to negative regulation to apo-ptosis or programmed cell death [38]. It has been considered tobe an important factor that provides potent resistance againstchemotherapeutic drugs [39]. Recent studies have demonstratedthat survivin is correlated with osteosarcoma and could be usedas a molecular marker for prognosis of osteosarcoma [40,41]. Thereported techniques for gene detection included flow cytometry(FCM) [42], fluorescence in situ hybridization (FISH) [43], chro-mosome analysis [44], real-time quantitative reverse transcriptionpolymerase chain reaction (RT-PCR) [45]. However, these methodsare subjected to such limitations as time consuming, poor precision,and expensiveness. Therefore, there is a real need for developmentof highly sensitive and selective DNA biosensors for detection ofsurvivin gene remains challenging.

In this study, a novel electrochemical DNA biosensor based ongraphene-three dimensional (3D) nanostructure gold nanocom-posite modified glassy carbon electrode (G-3D Au/GCE) isfabricated for detection of survivin gene in osteosarcoma. TheG-3D Au nanocomposite is prepared by a one-step electrochemi-cal co-deposition technique. A “sandwich-type” detection strategyis employed to fabricate the electrochemical DNA biosensor,namely, the sulfhydryl (SH) modified capture probe is first self-assembled at the G-3D Au nanocomposites by Au S bond. In thepresence of target DNA, the capture probe and reporter probeflanked the target sequence to form the sandwich sensing mode.Streptavidin–horseradish peroxidase (HRP) connected with biotinlabeled at the end of reporter probe via streptavidin–biotin affinitybinding offers an enzymatically amplified electrochemical currentsignal for detection of target DNA. Under optimal experimentalconditions, the DNA biosensor showed excellent sensitivity andselectivity and has been successfully used for assay of real-life PCRsamples with a satisfactory result.

2. Experimental

2.1. Chemicals and materials

Natural powder graphite (sized ≤ 30 �m) is purchased from

tetrahydrate (HAuCl4·4H2O, 47.8% Au), Tris-(hydroxymethyl)aminomethane, nitric acid, sulfuric acid, potassium perman-ganate, ethanol are provided by Sinopharm Chemical Reagent

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o., Ltd. (China). Ethylenediaminetetraacetic acid (EDTA), mer-aptohexanol (MCH) and bovine serum albumin (BSA) arerom Sigma–Aldrich (St. Louis, MO, USA). Tris (2-carboxyethyl)hosphine hydrochloride (TCEP) is obtained from Shanghaiangon Biological Engineering Technology Services Reagents Co.,td. (China). TMB substrate (TMB = 3,3′,5,5′-tetramethylbenzidine;eogen K-blue low activity substrate) is purchased from Neogen

USA). Streptavidin–HRP (horseradish peroxidase) is supplied fromoche Diagnostics (Mannheim, Germany). All the chemicals are ofnalytical reagent grade and used without further purification.

All the synthetic oligonucleotides are obtained from TaKaRaiotechnology (Dalian, China) Co., Ltd. Their base sequences are

isted as follows: DNA capture probe (30-base sequence S1)-5′-SH-GC CCT TCT TGG AGG GCT GCG CCT GCA CCC-3′; DNA reporterrobe (30-base sequence S2)-5′-AGG ACC ACC GCA TCT CTA CATCA AGA ACT-3′; target DNA (60-base sequence S3)-5′-AGT TCTGA ATG TAG AGA TGC GGT GGT CCT GGG TGC AGG CGC AGC CCTCA AGA AGG GCC-3′; single-mismatched DNA (60-base sequence4)-5′-AGT TCT TGA ATG TAC AGA TGC GGT GGT CCT GGG TGC AGGGC AGC CCT CCA AGA AGG GCC-3′; noncomplementary DNA (60-ase sequence S5)-5′-ACC ACG TGG CCA GTG GCG CCG GGG AGGAG CCA TTG AGA CCC AGA GCA GCA GTT CTG AAG-3′). Buffersor hybridization measurement are TMB substrate. Hybridizationuffer is 10 mM PBS buffer containing 0.5 M NaCl (pH 7.4). DNA

mmobilization buffer: 10 mM Tris-HCl containing 1 mM EDTA,0 mM TCEP and 1 M NaCl (pH 7.4). Washing buffer is 10 mM PBSuffer containing 0.1 M NaCl (pH 7.4). Enzyme diluent is 0.01 M PBSuffer with 2% BSA (pH 7.4). All solutions are prepared with Milli Qater (18 M� cm resistivity) from a Millipore system.

.2. Apparatus

All electrochemical measurements were performed on a CHI60D Electrochemical Workstation (CH Instrument, USA) with

three-electrode system consisting of glassy carbon electrodeGCE, diameter: 3 mm), an Ag/AgCl (with saturated KCl) referencelectrode and a platinum wire auxiliary electrode. Electrochem-cal impedance measurements were carried out in a 0.1 M KClolution containing 10 mM K3[Fe(CN)6] + 10 mM K4[Fe(CN)6] (1:1)n a frequency range from 0.05 to 105 Hz, the amplitude was.0 mV. Cyclic voltammetry (CV) is carried out at a scan ratef 100 mV s−1. Amperometric detection is performed at a fixedotential of 100 mV. Steady-state currents are recorded within00 s. Scanning electron microscopic (SEM) images and energy-ispersive X-ray spectroscopy (EDS) are collected on a Hitachi-4800 scanning electron microscopy (Tokyo, Japan) equipped withn energy-dispersive X-ray spectroscopy detector.

.3. Preparation of graphite oxide

Graphite oxide is synthesized from natural powder graphite by aodified Hummers’ method [46,47]. Briefly, graphite powders are

rst oxidized by potassium permanganate in the presence of con-entrated nitric acid and sulfuric acid for 30 min. After oxidationf graphite, the mixture is added to excess water, washed with a% HCl aqueous solution, and then repeatedly washed with water

ntil the pH of filtrate reached neutral. The as-synthesized GO isuspended in water to give a brown dispersion, which is dialyzedor one week to completely remove residual salts and acids. Exfo-iated graphite oxide (exfoliated GO) is obtained by ultrasound ofhe 1.0 mg mL−1 GO dispersion using a sonifier. The obtained brownispersion is then centrifuged at 3000 rpm for 5 min to remove anynexfoliated graphite oxide (an extremely small amount).

a Acta 767 (2013) 50– 58

2.4. Preparation of G-3D Au/GCE

Glassy carbon electrode (GCE) is polished successively with 1.0,0.3 and 0.05 �m alumina powder to form a smooth, shiny sur-face. Then it is cleaned ultrasonically in 1:1 HNO3, ethanol andMilli-Q water for 1 min, respectively, and dried with blowing N2.8 �L exfoliated GO suspension is spread on a pretreated bare GCEusing a micropipette tip. The film is dried in a vacuum desicca-tor. The GO-coated electrode is immersed in 2.8 mM HAuCl4 and0.1 M H2SO4 solution and a one-step electrochemical co-reductionis performed by cyclic voltammetry (CV) in a potential range from0.0 to −1.5 V. The resultant G-3D Au/GCE is sonicated in deion-ized water and electrochemically cleaned by cycling the electrodepotential between −0.35 and 1.5 V at 0.1 V s−1 in 0.5 M H2SO4 solu-tion. The real surface of the clean electrode is calculated from thecyclic voltammogram in 0.5 M H2SO4 solution.

2.5. DNA self-assembly at G-3D Au/GCE surfaces

The cleaned G-3D Au/GCE is incubated in an immobilizationbuffer containing 10 �M capture probes modified with thiolate for2 h at room temperature. The single-stranded DNA (ssDNA) modi-fied electrode is then treated with 2.5 mM mercaptohexanol (MCH)for 1 h to obtain a well-aligned ssDNA/MCH. Prior to hybridiza-tion reaction, the modified electrode is blocked by 5% (w/v) bovineserum albumin (BSA) for 15 min to prevent nonspecific adsorptionof biotin labeled at the end of reporter probe.

2.6. Hybridization with target DNA

The synthetic targets or the PCR amplicons are first mixedwith the biotinylated reporter probe (100 nM) in the hybridiza-tion buffer. The PCR real samples are heated at 95 ◦C for 10 minand then cooled in ice bath before use. The sensor surface modi-fied with capture probes is incubated in the hybridization buffercontaining biotinylated reporter probes and target DNA at 37 ◦C for1 h. After rinsing with ishing buffer, the sensor is blocked by 5% BSAfor 1.5 h and then incubated with 3 mL of avidin-HRP (0.5 U/mL) for15 min at room temperature. After removing any remaining, thesandwich structure is formed and can be used for electrochemicalmeasurements.

2.7. Electrochemical measurement

The electrochemical signal of the enzymatically produced TMBsubstrate was measured by cyclic voltammetry (CV) and ampero-metric I–t curve. CV was carried out at a scan rate of 100 mV s−1.Amperometric detection was performed at 100 mV. Steady-statecurrent was usually reached within 100 s.

3. Results and discussion

3.1. Characterization of G-3D Au/GCE

A schematic representation of the present sensor fabricationprocedure is illustrated in Scheme 1. The G-3D Au/GCE was firstprepared by a one-step electrochemical co-reduction strategy andthen used as the substrate for DNA immobilization and hybridiza-tion. In order to obtain a 3D gold nanostructure, the potentialwindow in CV was set between 0.0 and −1.5 V to promote rig-orous hydrogen liberation. The hydrogen bubbles were used as

dynamic template for deposition of three-dimensional gold struc-tures [48–50]. The electrochemical reduction process was shown inFig. S-1. As shown in Fig. S-1, the large reduction current in the firstcycle should be due to the reduction of the surface oxygen groups

A.-L. Liu et al. / Analytica Chimica Acta 767 (2013) 50– 58 53

DNA b

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gfttotTopffoadtod

tiab

Scheme 1. Schematic representation of the fabrication procedure of

f GO since the reduction of water to hydrogen occurs at more neg-tive potentials, then the reduction current decreased considerablyith the increasing potential scans and disappeared after severalotential scans, demonstrating that the GO was reduced electro-hemically to ERGO. Scanning electron microscope was used toxamine the surface morphologies of bare GCE, Au-nanoparticleodified GCE (Au/GCE), GO/GCE, ERGO/GCE and G-3D Au/GCE

Fig. 1). Fig. 1A shows a smooth surface morphologie of bare GCE.n Fig. 1B, the spherical structures correspond to Au nanoparticlesispersed on the GCE surface. The SEM images of GO/GCE (Fig. 1C)nd ERGO/GCE (Fig. 1D) reveal the typically crumpled and wrin-led GO and graphene sheets structure. The SEM image of a G-3Du/GCE in Fig. 1E shows pinecone-like 3D gold nanostructures andraphene sheets. The TEM images of GO, ERGO and G-3D Au washown in Fig. S-2. As shown in Fig. S-2C, nanogold particles in thecale of about 50–180 nm were deposited on the graphene flakes.he pinecone-like 3D gold nanostructures in Fig. 1E were composedf those nanogold particles. Energy–dispersive X-ray spectroscopyEDS) analysis of the 3D nanostructures (Fig. 1F) clearly shows Cnd Au peaks, indicating the successfully deposition of gold nano-tructures on graphene sheets.

The formation mechanism of pinecone-like 3D nanostructuredold can be explained as follows: the deposited gold atoms firstormed nuclei at the defects available at the graphene surface dueo the fast electron transfer of edge–plane-like defects [51,52]. Thenhe hydrogen bubble evolution arising from the reduction of H+

ccurs on the surface of gold nuclei and acts as a dynamic templateo assist the orientation growth of gold nanocrystal [28,48–50].he subsequent growth of gold crystals would preferentially occurn the preformed gold nuclei through a surface reaction, which isossibly due to the relatively high activation energy for the sur-ace reaction [53]. Thus, pinecone-like 3D nanostructured gold wasormed. Based on the coulombic integration of the reductive peakf gold oxide at 0.9 V in the cyclic voltammogram of 0.5 M H2SO4 at

G-3D Au/GCE shown in Fig. 2A, the electroactive surface area ofeposited gold nanostructures was estimated to be 3.8 times largerhan that of Au deposited on a GCE and 8.8 times larger than thatf a planar Au electrode with the same geometric area (0.07 cm2,iameter: 0.15 cm) [54].

Fig. 2B shows the cyclic voltammograms of the prepared elec-

rodes in 0.5 M H2SO4. A redox pair (peaks I and peaks II in thenset) of phenolic hydroxyl groups on the ERGO/GCE appears. Such

redox pair was also observed in the CV of the G-3D Au/GCEesides the characteristic peaks for Au at 1.2 V and 1.4 V [55]. These

iosensor based on graphene-3D nanostructure gold nanocomposite.

phenomena indicate that graphene and Au are simultaneouslyformed on the GCE in the present codeposition technique.

The [Fe(CN)6]3−/4−couple is widely used as an electrochem-ical probe to evaluate the characteristics of electrochemicalactive materials [56]. Fig. 2C shows the cyclic voltammogramsof [Fe(CN)6]3− at differently modified electrodes in 10 mM[Fe(CN)6]3− and 0.1 M KCl solution at 100 mV s−1. A pair of redoxpeaks is observed in all CV traces. The current response of theGO modified GCE (GO/GCE) toward [Fe(CN)6]3−/4− is the lowestbecause of the insulating property of GO. After electrochemicalreduction of GO, the redox peak currents for ferric cyanide probeon the ERGO/GCE are 13% higher than that at the correspondingbare electrode. This enhancement is attributed to the excellent con-ductivity, large surface area, and remaining oxygen-related defectsof the ERGO film [9,57,58]. In addition, it is clear that the redoxpeak currents of the G-3D Au/GCE are the highest among the mod-ified electrodes (about 25% higher than that at the correspondingbare electrode), indicating that the electrochemical activity of G-3Dnanostructured gold nanocomposite was further enhanced by theco-deposited gold nanostructures.

3.2. Characterization of the biosensor based on G-3D Au/GCE

The preparation process of electrochemical biosensor is charac-terized by electrochemical impedance spectroscopy (EIS). Fig. 3Bshows Nyquist plots for stepwise modification of G-3D Au/GCE. Forcomparison, the Nyquist plot for a bare flat Au electrode (Fig. 3A)is also presented. The semicircle portion at high frequencies corre-sponds to the charge transfer limiting process. The charge transferresistance Ret can be directly measured as the semicircle diameter.For the bare Au electrode, the Ret is 127.4 �. After immobilizationof thiolated capture probe with negative charges on its phosphatebackbone, the Ret increases from 127.4 to 4965.0 �. The increasein Ret is due to the immobilization of negatively charged captureprobe monolayer on the electrode surface which repels the accessof electrochemical probe carrying negative charges to exchangeelectrons with the substrate electrode via the electrostatic inter-actions. When MCH is employed to remove the nonspecificallyadsorbed probe, the Ret decreases from 4965.0 to 4635.0 �. Thiscan be attributed to less negative charges on the electrode sur-

face after removal of the nonspecifically adsorbed DNA by MCH.Hybridization of target DNA with the capture probe on the modi-fied electrode surface will increase negative charges, resulting moreresistance to charge transfer of the electrochemical probe with

54 A.-L. Liu et al. / Analytica Chimica Acta 767 (2013) 50– 58

GO/G

t7pfbf

GtG

Fig. 1. SEM images of (A) bare GCE, (B) Au/GCE, (C) GO/GCE, (D) ER

he substrate electrode, thus an increase in Ret from 4635.0 to955.0 � is observed. After the affinity conjugation of the capturerobe/target/reporter probe modified electrode with avidin-HRPor 15 min, the Ret increases from 7955.0 to 14,000 �, due tolockage of the bulky avidin-HRP molecules on the electrode sur-ace.

As shown in Fig. 3B, when exfoliated GO was modified onto aCE surface, the value of Ret increased as compared to the bare GCE,

he Ret increases from 96.5 to 342.0 � indicating that the exfoliatedO acted as an insulating layer which made the interfacial charge

CE, (E) G-3D Au/GCE and EDS spectrum of G-3D Au on the GCE (F).

transfer difficult. After electrochemical co-reduction of the exfo-liated GO with HAuCl4, the Ret decreased distinctively to 37 �,suggesting that graphene-3D nanostructure gold nanocompositehad accelerated electron transfer between the electrochemicalprobe [Fe(CN)6]3−/4− and the electrode. Even after probe immo-bilization and the following modification steps, the Ret values were

also quite low compared with corresponding steps for flat goldelectrode (Fig. 3B), showing that ERGO-Au/GCE presented highelectrical conductivity due to the conductive graphene sheets con-necting 3D nanostructure gold [9].

A.-L. Liu et al. / Analytica Chimica Acta 767 (2013) 50– 58 55

-0.5 0.0 0.5 1.0 1.5

-800

-600

-400

-200

0

200

400

600

(a)

(c)

Potential Vs. Ag/ AgCl / V

(A)

-0.2 0.0 0.2 0.4 0.6 0.8-160

-120

-80

-40

0

40

80

120

160

GO/ GCE

ERGO-Au/ GCEERGO/ GCE

Cu

rre

nt

/ µA

Cu

rre

nt

/ µA

Potential Vs. Ag/ AgCl / V

bare GCE

(C)

-0.5 0.0 0.5 1.0 1.5

-800

-600

-400

-200

0

200

400

Potential/ V(Vs.Ag/ AgCl)

Potential/V(Vs. Ag/ AgCl)

0.0

I

II

G 3D-Au/ GCE

ERGO/GCE

GO/GCE

GCE

GCE

GO/GCE

(B)

0.7 1.4

-90

0

90

Cu

rre

nt

/ µA

Cu

rre

nt

/ µA

F (b)) a0 nd (C)

3b

odterhcDbi

FpA(

ig. 2. (A) Cyclic voltammograms (CVs) of G-3D Au/GCE (curve (a)), Au/GCE (curve.5 M H2SO4. (B) CVs of different modified electrodes in a solution of 0.5 M H2SO4 a

.3. Electrochemical responses of the nanocomposite-based DNAiosensor

Fig. 4 shows the CV and I–t curves of capture probe (ssDNA)r capture probe/target DNA/reporter probe modified biosensor atifferent electrodes in TMB solution. As shown in Fig. 4A, there arewo pairs of redox peaks at the surface of ssDNA modified flat goldlectrode exposed in TMB substrate (curve a), these two pairs rep-esented the oxidation of TMB and the reduction of H2O2. Afterybridization with target DNA and reporter probe, a prominent

atalytic reduction peak is found in CV of capture probe/targetNA/reporter probe modified flat gold electrode (Fig. 4A, curve). This indicates that upon hybridization, the sandwich structure

s formed, and Streptavidin–HRP could conjugate with the biotin

0 6000 12000

0

2000

4000

6000

(e)

-Z

"/o

hm

Z'/ohm

(a)

Rs

Ret

CPE

Zw

(A)

ig. 3. (A) Impedance spectras (Nyquist plots) of bare flat gold electrode (AuE) with the sarobe/target DNA/reporter probe/MCH/AuE (d) and capture probe/target DNA/reporteru/GCE (a), bare GCE (b), GO/GCE (c), capture probe/MCH/G-3D Au/GCE (d), capture pro

f) and capture probe/target DNA/reporter probe/avidin-HRP/MCH/G-3D Au/GCE (g).

nd planar gold electrode with the same geometric area (curve (c)) in a solution of CVs of different modified electrodes in 10 mM [Fe(CN)6]3− and 0.1 M KCl solution.

labeled at the end of reporter probe via affinity reaction. NotablyHRP does not directly exchange electron with the electrode becauseits redox site is shielded within insulating peptide backbones [59].Therefore, small redox molecule TMB, as an electron shuttle, candiffuse in and out of the redox site of HRP. And the catalytic reduc-tion of H2O2 coupling with the redox reaction of TMB occurs on theelectrode [59,60]. Thus, the HRP modified on the surface of goldelectrode could efficiently catalyze reduction reactions of H2O2with the redox reaction of TMB, leading to significant amplifica-tion of reduction current [60,61]. However, due to absence of the

biotinylated reporter probe, which could conjugate the HRP, thiscatalytic reduction current on the ssDNA modified biosensor isnot obvious. Accordingly, the sandwich-mode biosensor can dis-criminate ssDNA and ssDNA/target DNA/reporter probe complex

0 500 1000 1500

0

200

400

(g)

- Z

"/o

hm

Z'/ohm

(a)

Rs

Ret

CPE

Zw

(B)

me geometric area (a), capture probe/MCH/AuE (b), capture probe/AuE (c), capture probe/avidin-HRP/MCH/AuE (e). (B) Impedance spectras (Nyquist plots) of G-3Dbe/G-3D Au/GCE (e), capture probe/target DNA/reporter probe/MCH/G-3D Au/GCE

56 A.-L. Liu et al. / Analytica Chimica Acta 767 (2013) 50– 58

0.0 0.2 0.4 0.6 0.8

-20

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E/V(Vs.Ag/ AgCl)

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-30

-20

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0

10

20

(b)

Cu

rre

nt/

µA

E/V(Vs. Ag/ AgCl)

(a)

(B)

0.0 0.2 0.4 0.6 0.8-80

-60

-40

-20

0

20

40

(b)

(a)

E/V(Vs. Ag/ AgCl)

(C)

Cu

rre

nt/

µ A

Cu

rre

nt/

µA

F trate.e

v(bdaati

3

trT(samg(pciptdibom

To further evaluate the detection performance of the graphene-3D nanostructured gold nanocomposite-based biosensor, theamperometric signals are measured before and after hybridization

0 20 40 60 80 100-40

-30

-20

-10

0

0

2

4

6

8

10

12

(e)(b) (d)(c)(a)

(e)Cu

rre

nt/

µA

Cu

rre

nt/

µA

Time(s)

(a)

Fig. 5. Current–time curves of background (e), a capture probe modified G-3D

ig. 4. CVs corresponding to AuE (A), Au/GCE (B) and G-3D Au/GCE (C) in TMB subslectrodes. Insets are the corresponding I–t curves.

ery well. The similar phenomenon is also observed at the Au/GCEFig. 4B, curves a and b) and G-3D Au/GCE (Fig. 4C, curves a and), but the reduction current signals are gradually enhanced. Theifference in reduction peak current in Fig. 4C (curves a and b) isbout 6.7 times and 3.2 times as large as that in Fig. 4A (curves and b) and Fig. 4B (curves a and b), respectively, indicating thathe graphene–Au nanocomposite modified GCE can significantlymprove the electrocatalytic activity.

.4. The selectivity of DNA electrochemical biosensor

Detection of target DNA is also investigated by amperometricechnique, which provides a rapid and direct measure of the cur-ent associated with the HRP catalyzed electrochemical process.he potential is held at the catalytic reduction potential for H2O2100 mV). A decay curve for current (I) vs time (t) rapidly reachesteady-state current within 100 s (Fig. 5). The selectivity of thisssay in discriminating complementary DNA (S3) from one baseismatched DNA (S4) and noncomplementary DNA (S5) is investi-

ated as shown in Fig. 5. It is clear that a large amperometric signal10.4 �A, Fig. 5, curve a) is obtained after hybridization with com-lementary DNA, which was more than one order of magnitudeompared with the background signal (0.227 �A, Fig. 5, curve e). Itndicates that capture probes show the excellent affinity for com-lementary target. In the presence of single base mismatched DNA,he amperometric signal (7.09 �A, Fig. 5, curve b) is significantlyecreased compared with the complementary DNA, demonstrat-

ng that the complete hybridization is not accomplished due to thease mismatch. Moreover, no significant difference in current isbserved for capture probe and its hybridization with noncomple-entary DNA (0.313 �A, 0.297 �A, Fig. 5, curves c and d), since no

(a) ssDNA modified electrodes and (b) ssDNA/target DNA/reporter probe modified

successful hybridization occurred due to the sequence mismatchbetween the probe and the noncomplementary sequence.

3.5. The sensitivity of fabricated DNA biosensor

Au/GCE (d) and after hybridization with the noncomplementary sequence (c), one-base mismatch sequence (b) and complementary target sequence (a). Inset showsthe bar graph of the amperometric currents when the capture probe hybridized withdifferent gene fragments. Error bars show the standard deviations of measurementstaken from at least three independent experiments.

A.-L. Liu et al. / Analytica Chimica Acta 767 (2013) 50– 58 57

0 20 40 60 80 100-15

-12

-9

-6

-3

0

(n)

Time(s)

(a)

(A)

-2 -1 0 1 2 3 4 5 6

0

2

4

6

8

10

12

-1.5 -1.0 -0.5 0.0 0.5 1.0

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Cu

rre

nt/

µA

Logarithmic DN A Concen tration / pM

Cu

rre

nt/

µA

Logarit hmic (DN A Concen tration / pM)

(B)

Cu

rre

nt/

µA

Fig. 6. (A) Amperometric measurements for the capture probe hybridized with synthetic target DNA at a series of concentrations (From a to n): 100 nM, 50 nM, 10 nM, 5 nM,1 tion cus ts and

watctDc3sobGd

3

pppg

Fctm

nM, 500 pM, 100 pM, 10 pM, 5 pM, 1 pM, 500 fM, 100 fM, 50 fM, 0 fM. (B) Calibraignal is the same as that in (A). Inset shows the linear relationship between curren

ith the target DNA at different concentrations. As shown in Fig. 6And B, the current increases with the increase of the concentra-ion of target DNA in a range from 50 fM to 100 nM. Under optimalonditions for hybridization, a good linear relationship betweenhe current signal and the logarithmic function of complementaryNA concentration (shown in Fig. 6B, inset) is observed in a con-entration range from 50 fM to 5.0 pM with a detection limit of.4 fM using 3� (where � is the standard deviation of the blankolution, n = 3). The reproducibility of the biosensor for detectionf 0.1 pM target DNA was 7.46% (n = 3). Compared with Au/GCE-ased biosensor and planar gold electrode-based biosensor, the-3D Au/GCE-based biosensor had lower detection limit for DNAetection (shown in Figs. S-3 and 4, and Table S-1).

.6. Detection of PCR products

All the PCR samples are obtained from the first affiliated hos-

ital of Fujian Medical University. The electropherograms of PCRroducts are shown in Fig. 7A. The PCR amplification products fromositive real samples present the light brands in the 1.5% agaroseel (Fig. 7A, lane 1). However, the PCR products from negative real

ig. 7. (A) The electropherogram of PCR products. The lanes from left to right: (1) posiurves of blank (PCR system without cDNA template) (c), ssDNA modified G-3D Au/GCE

he bar graph of the amperometric currents when the ssDNA modified G-3D Au/GCE heasurements taken from at least three independent experiments.

rve of currents vs logarithmic target DNA concentration, where the definition of logarithmic target DNA concentration (from 50 fM to 5 pM).

samples (Fig. 7A, lane 2) and blank background (Fig. 7A, lane 3) donot show any light brands in the gel.

Fig. 7B shows the amperometric signals obtained when the cap-ture probe for survivin is immobilized and hybridized with differentreal samples in the hybridization step. The amperometric signalsobtained from the hybridization of the capture probe with posi-tive and negative real samples give mean average of 2220.6 and510 nA, respectively (shown in Fig. 7B, inset). The averaged currentfor blank background is 410 nA. The obvious increase in the mag-nitude of amperometric signal obtained with positive real samplesis observed. The increase of amperometric signal indicates that thehybridization of the capture probe with positive real samples isoccurred at the surface of G-3D Au/GCE, and the Streptavidin–HRPhas fixed onto ds-DNA via biotin–avidin affinity bond. If the bloodsample is negative, the amplified PCR product would not containa target sequence complementary to the specific capture probe.Thus, the hybridization would not occur between the negative real

sample and the immobilized capture probe, and the amperomet-ric signal is nearly as high as the background. The results obtainedfrom the electrochemical DNA biosensor are in good agreementwith those of the gel electrophoresis.

tive real sample (2) negetive real sample (3) blank background. (B) Current–timehybridized with negative real sample (b) and positive real sample (a). Inset showsybridized with different real samples. Error bars show the standard deviations of

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8 A.-L. Liu et al. / Analytica C

. Conclusion

In summary, a green, simple and fast electrochemical approachor the synthesis of graphene-3D nanostructure gold nanocom-osite film is developed by using an electrochemical codepositionechnique. Due to the high conductivity, electrocatalysis, biocom-atibility and large active surface of the resultant nanocomposite,he present sandwich-mode amperometric DNA biosensor exhib-ted excellent sensitivity and selectivity for detection of survivinene. This new biosensor for assay of complementary sequencen the PCR amplified real samples shows satisfactory results.urthermore, the electrochemical properties of graphene-3D nano-tructure gold composite are expected for further extensivepplications in protein, enzyme biosensors.

cknowledgments

The authors gratefully acknowledge the financial supportf the National High Technology and Development of China2012AA022604), the Nationnal Natural Science Foundation ofhina (20975021, 21175023, 81171668, 21275028), the Fujianrovincial University-Industry Cooperation Sciecne & Technologyajor Program (2010Y4003), the Foundation of Fujian Key Labo-

atory of Hematology (2009J1004), the Scientific Research Majorrogram of Fujian Medical University (09ZD013), and the Naturalcience Foundation of Fujian Province of China (2010J06011) andoundation of Fujian Provincial Department of Education (JA10126,A11110, JA12130).

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.aca.2012.12.049.

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