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Biosensors and Bioelectronics 31 (2012) 399– 405

Contents lists available at SciVerse ScienceDirect

Biosensors and Bioelectronics

j our na l ho me page: www.elsev ier .com/ locate /b ios

ovel electrochemical catalysis as signal amplified strategy for label-freeetection of neuron-specific enolase

ing Han, Ying Zhuo ∗, Ya-Qin Chai, Ya-Li Yuan, Ruo Yuan ∗

ducation Ministry Key Laboratory on Luminescence and Real-Time Analysis, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China

r t i c l e i n f o

rticle history:eceived 29 July 2011eceived in revised form 25 October 2011ccepted 27 October 2011vailable online 25 November 2011

eywords:mmunosensorlectrochemical catalysisold nanoparticles functionalized graphene

a b s t r a c t

A label-free electrochemical immunoassay for neuron-specific enolase (NSE), a kind of lung cancermarker, was developed in this work via novel electrochemical catalysis for signal amplification. The newamplified strategy was based on the electrochemical catalysis of nickel hexacyanoferrates nanoparticles(NiHCFNPs) in the presence of dopamine (DA). NiHCFNPs, which were assembled on the porous goldnanocrystals (AuNCs) modified glassy carbon electrode (GCE), could exhibit a distinct pair of redox peakscorresponding to anodic and cathodic reactions of hexacyanoferrate (II/III). Subsequently, gold nanopar-ticles functionalized graphene nanosheets (Au–Gra) were coated on the surface of NiHCFNPs/AuNCs film.Then an enhanced amount of neuron-specific enolase antibody (anti-NSE) could be loaded to obtain a sen-sitive immunosensor of anti-NSE/Au–Gra/NiHCFNPs/AuNCs/GCE due to the strong adsorption capacity

anosheets (Au–Gra)ickel hexacyanoferrates nanoparticles

NiHCFNPs)opamine (DA)euron-specific enolase (NSE)

and large specific surface area of Au–Gra. More importantly, the oxidation peak current can be enor-mously enhanced towards the electrocatalytic oxidation of DA based on NiHCFNPs, resulting in thefurther improvement of the immunosensor sensitivity. Under optimal conditions, the electrochemicalimmunosensor exhibited a linear range of 0.001–100 ng/mL with a detection limit of 0.3 pg/mL (S/N = 3).Thus, the proposed immunosensor provides a rapid, simple, and sensitive immunoassay protocol for NSEdetection, which may hold a promise for clinical diagnosis.

. Introduction

Nowadays, cancer is one of the most threatening diseases foruman beings. The quantitative detection of tumor biomarkers,

ncluding serum tumor markers and potential prognostic factors,lays an important role in clinical early diagnosis (Faraggi andramar, 2000). Neuron-specific enolase (NSE) is a sensitive, specific,nd reliable tumor markers for small-cell lung carcinoma (SCLC) athe time of diagnosis (Oremek et al., 2007; Erbaycu et al., 2010). Ashe �-subunit of enolase, NSE presents primarily in the cytoplasm ofeurons and neuroendocrine cells. The NSE levels are 5–12 ng/mL inerum and 20 ng/mL in cerebrospinal fluid in normal human beingsMarangos and Schmechel, 1987). Therefore, the determination ofSE level is of great importance to process the early diagnostic andrognostic values for monitoring the SCLC state.

Electrochemical immunosensors, based on the specificntigen–antibody recognition, have gained wide interest in

etection and quantification of bio-molecules (Wu et al., 2007;rruda et al., 2009). Recently, increasing interests have been

ocused on label-free amperometric immunosensor due to their

∗ Corresponding authors. Tel.: +86 23 68252277; fax: +86 23 68252277.E-mail addresses: yingzhuo@swu.edu.cn, yingzhuo@126.com (Y. Zhuo),

uanruo@swu.edu.cn (R. Yuan).

956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2011.10.055

© 2011 Elsevier B.V. All rights reserved.

rapid recognition, simple fabrication and operation (Okuno et al.,2007; Mun et al., 2010). However, the sensitivity has been limitedas the lack of signal amplified strategy for most label-free amper-ometric immunosensors. Lately, there are some reports of thelabel-free amperometric immunosensors based on enzyme biocat-alytic amplified strategy (Zhuo et al., 2006; Liu et al., 2011), but thedefect of irreversible enzyme inactivation and relatively expen-sive of enzyme protein restricts the further application. Nickelhexacyanoferrate nanoparticles (NiHCFNPs) have been reportedto be a versatile nanomaterial for the label-free immunosensorconstruction because of their well-defined and reproducibleredox voltammetric responses. Furthermore, as a kind of smallbiomolecule with redox activity, it was reported by Prabhu et al.(2011) dopamine (DA) could be electrocatalytic oxidated by metalhexacyanoferrate. Thus, a new amplified strategy based on theelectrochemical catalysis of NiHCFNPs in the presence of DA wasdeveloped for immunosensor construction in this work.

Biomolecular immobilization has been considered as one of themost important points in biosensor fabrication. Au nanoparticleshave been recognized as the ideal nanomaterials for biomolecularimmobilization as they could adsorb many biomolecules and main-

tain their bioactivities (Ding et al., 2004; Wei et al., 2007; Shanet al., 2010). Recently, an explosion of interest has been focusedon graphene (Gra) which possesses many extraordinary proper-ties (Kaner, 2008; Geim, 2009; Wang et al., 2010), including good

400 J. Han et al. / Biosensors and Bioelectronics 31 (2012) 399– 405

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cheme 1. Illustration of the stepwise immunosensor fabrication process: (a) formanti-NSE loading; (e) blocking with 0.25% BSA; (f) incubation with NSE. (A) TEM imC) CV measurements of current response with different concentrations of NSE in th

echanical properties, large surface area, an excellent electronransfer rate for extensive applications in sensors, electrochemi-al devices, polymer nanocomposites and so on (Ramanathan et al.,008; Xia et al., 2010; Huang et al., 2011). Therefore, we take advan-age of dual-effects of gold nanoparticles and graphene nanosheetso prepare the gold nanoparticles functionalized graphene sheetsAu–Gra) nanohybrid, which exhibited an enhanced adsorption forntibody immobilization (Han et al., 2011).

In this work, a novel electrocatalytic strategy was developed forhe fabrication of a label-free NSE immunosensor. The large surfacerea of Au–Gra can increase the amount of anti-NSE loading and theood conductivity of Au–Gra can also enhance the electroactivity ofiHCFNPs. NiHCFNPs are one of the excellent electroactive nano-aterials and favorably enhance the oxidation peak current by the

lectrochemical catalysis of DA. Experiment results show that themperometric response of the immunosensor could be amplifiedhich can enormously improve the sensitivity of immunosensor.

he details of the attractive response performances of the pro-osed immunosensor and potential merits for NSE detection areubstantiated as follows.

. Experimental

.1. Reagent and materials

NSE (0–100 ng/mL) and NSE monoclonal antibody (anti-NSE)ere purchased from Advanced Life Science Institute, Inc., Saitama,

apan. Graphene oxide sheets (GO) were obtained in Pioneeranotechnology Co. (Nanjing, China). DA was purchased fromhemical Reagent Co. (Chongqing, China). Bovine serum albu-in (BSA, 96–99%), gold chloride (HAuCl4), sodium citrate and

-ascorbic acid (AA) were obtained from Sigma Chemical Co.St. Louis, MO, USA). K3Fe(CN)6 and NiCl2·6H2O were obtainedrom Chemical Reagent Co. (Sichuan, China). All other materi-ls used were of the highest quality available and purchasedrom regular sources. Bi-distilled water was used throughout thistudy. Phosphate buffered solutions (PBS) (pH 7.4) were pre-ared using 0.1 mol/L Na2HPO4, 0.1 mol/L KH2PO4, and 0.1 mol/LaCl.

.2. Apparatus

The cyclic voltammetric (CV) measurements were carried outith a CHI 660D electrochemical workstation (Shanghai Chen

f AuNCs film; (b) assembled the NiHCFNPs; (c) formation of Au–Gra monolayer; (d) Au–Gra. (B) Electrocatalytic oxidation mechanism of DA at the prepared electrode.sence of DA.

Hua Instrument, Co., China). The AC impedance of the immuno-electrode membrane was measured with a Model IM6e (ZAHNERElektrick, Germany). A three-electrode electrochemical systemwas composed of a platinum wire as the auxiliary electrode,a saturated calomel electrode (SCE) as the reference electrodeand a modified glassy carbon electrode (GCE, ˚ = 4 mm) as theworking electrode. The topograghs of the Au substrates mod-ified with different materials were investigated with atomicforce microscopy (AFM, Veeco, Woodbury, NY, USA). The sizeof gold nanoparticles functionalized graphene nanosheets wasestimated from transmission electron microscopy (TEM) (H600,Hitachi Instrument, Japan). The pH measurements were made witha pH meter (MP 230, Mettler-Toledo Switzerland) and a digi-tal ion analyzer (Model PHS-3C, Dazhong Instruments, Shanghai,China).

2.3. Preparation of NiHCFNPs

The NiHCFNPs were prepared as following procedure accord-ing to the literature (Yang et al., 2006). Briefly, 40 mL of 0.01 mol/LNiCl2 aqueous solution was first dropped gradually into 40 mL of0.05 mol/L K3Fe(CN)6 solution containing 0.05 mol/L KCl under stir-ring. Then the resulting mixture solutions were vigorously agitatedfor 5 min, and immediately centrifuged, washed with double dis-tilled water for three times. Subsequently, the NiHCFNPs weredried in a vacuum at room temperature and gave a powered sub-stance.

2.4. Preparation of Au–Gra

Au–Gra were prepared by following steps: At first, GO were dis-solved in water by ultrasonic dispersion. And then 0.1 g AA wasadded to 10 mL of an aqueous dispersion of GO (1 mg/mL) andstirred for overnight at room temperature. Next, 2 mL of 1% goldchloride solution was added to the above mixture and stirred for 8 h.Following that, in order to remove the excessive graphite oxide andAA, the product of Au–Gra were centrifugally washed extensivelywith double distilled water and finally dispersed in bi-distilledwater. Herein, the Au–Gra were characterized by TEM, which indi-

cated the gold nanoparticles were integrated uniformly with thegraphene nanosheets by one step chemical method of reducing GOand HAuCl4 via AA under gentle conditions. And TEM of Au–Gra ispresented in Scheme 1(A).

Bioelectronics 31 (2012) 399– 405 401

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J. Han et al. / Biosensors and

.5. Fabrication of proposed immunosensor

To obtain mirror-like surface, the GCE ( ̊ = 4 mm) was firstlyolished successively with 0.3 and 0.05 �m alumina powder. Andhen it was rinsed with distilled water and ethanol in ultrasonicath to remove the physically absorbed substance. After that, theCE was allowed to dry at room temperature.

Firstly, the pretreated GCE was immersed in 2 mL of 1% HAuCl4olution for electrochemical deposition under constant potentialf −0.2 V for 30 s to obtain porous gold nanocrystals (AuNCs) filmodified electrode (AuNCs/GCE). Then, 10 �L prepared NiHCFNPs

queous solution was dropped on the surface of AuNCs layer asedox probe via the interaction of –CN groups of NiHCFNPs anduNCs. Next, 10 �L Au–Gra was coated on NiHCFNPs membraneodified electrode. Following that, the obtained electrode was

ncubated in 0.5 mL anti-NSE solution at 4 ◦C for 12 h. To block theossible remaining active sites and avoid the non-specific adsorp-ion, 20 �L of 0.25% BSA solution was dropped on the electrode for.5 h at 37 ◦C. After every step, the modified electrode was thor-ughly cleaned with bi-distilled water to remove the physicallybsorbed species. The finished immunosensor was stored at 4 ◦Chen not in use. Scheme 1 may illustrate self-assembly proce-ure and the mechanism of the proposed electrochemical catalysistrategy.

.6. Experimental measurements

The electrochemical properties of the modified electrode wereharacterized by CV and EIS. The CV scan was taken from 0 to.8 V at 100 mV/s in PBS (PH 7.4). EIS measurements were done

n the presence of a 5.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixtures a redox probe at the formal potential of 220 mV. The alterna-ive voltage is 10 mV and the frequency range is from 1 × 10−2

o 1 × 106 Hz. The detection is based on the change of the anodiceak current (�I) response before and after the antigen–antibodyeaction, due to the immunocomplex hindering the access of redoxrobe to electrode. When the background current was stabilized,he anodic peak current response was recorded as I0. Then, themmunoreaction was carried out, and after that the anodic peakurrent response was recorded as I. The change of the �I was giveny �I = I0 − I.

. Results and discussion

.1. Characteristics of the immunosensor

The surface topographic feature of the self-assemble proce-ure of the immunosensor was investigated via AFM. The imagesf different modified gold surfaces are shown in Fig. 1. Fig. 1Andicates an image of the AuNCs film by electrodeposition, whichisplays a quite uniform triangle-like structure. Fig. 1B exhibits

topograph with globular features ascribed to the uniform dis-ribution of NiHCFNPs in AuNCs film by covalent bonds andlectrostatic interaction. After Au–Gra were assembled onto theesulting NiHCFNPs/AuNCs film, many nano-Au particles wrappedith a layer of thin film of Gra with the typical crumpled andrinkled structure was obtained (Fig. 1C). Such a hybrid mono-

ayer could present a large specific surface area, high surfaceree energy, good conductivity and a biocompatible environment.ubsequently, the anti-NSE adsorbed on Au–Gra/NiHCFNPs/AuNCs

urface was observed from Fig. 1D. The morphology exhibited anmage of a dense protein layer and a compact sheet-shaped struc-ure due to the presence of Au–Gra, indicating that the anti-NSEas successfully absorbed onto the electrode.

Fig. 1. AFM images of differently modified Au substrates: (a) AuNCs;(b) NiHCFNPs/AuNCs; (c) Au–Gra/NiHCFNPs/AuNCs; (d) anti-NSE/Au–Gra/NiHCFNPs/AuNCs.

402 J. Han et al. / Biosensors and Bioele

2000150010005000

0

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-1000Z im

(Ω)

Zre ( Ω)

abc

d

e

f

g

Fig. 2. EIS of different electrodes in 5.0 mmol/L K3[Fe(CN)6]/K4[Fe(CN)6](1:1) mixture: (a) bare GCE; (b) AuNCs/GCE; (c) NiHCFNPs/AuNCs; (d)Au–Gra/NiHCFNPs/AuNCs; (e) anti-NSE/Au–Gra/NiHCFNPs/AuNCs; (f) BSA/anti-NTS

3m

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SE/Au–Gra/NiHCFNPs/AuNCs; (g) NSE/BSA/anti-NSE/Au–Gra/NiHCFNPs/AuNCs.he frequency range is at 1 × 10−2 to 1 × 106 Hz at 25 ◦C (Zim vs. Zre at 220 mV vs.CE).

.2. Electrochemical impedance spectroscopy (EIS) of theodifying process

EIS is an effective method for studying the interface properties ofurface-modified electrodes and the electron-transfer resistance athe electrode surface. It is well known that the semicircle diameterf EIS is equal to Ret in the Nyquist diagram. And the Nyquist dia-rams at different modified electrodes in 5.0 mmol/L Fe(CN)6

3−/4−

olution are presented in Fig. 2. The bare GCE exhibits a small semi-ircle in the high frequency section (curve a). When the electrodeeposited with HAuCl4, the Nyquist diagram is approximately atraight line (curve b), indicating porous AuNCs film has excel-ent conductive properties, which formed a network like structurend constructed larger effective surface area than bare GCE surface.hen NiHCFNPs were loaded on the AuNCs/GCE, the Ret increased

litter but still less than the bare GCE (curve c). The reason may behat the microstructure of NiHCFNPs formed some barrier obstruct-ng the electron transfer. Next, the Ret decreased obviously whenu–Gra were assembled on the NiHCFNPs surface (curve d), due to

he Au–Gra could provide a large surface area and act as a conduct-ng tunnel to promote the electron transfer. When anti-NSE wasmmobilized onto the modified electrode, the Ret increased dra-

atically (curve e), which is ascribed to the inhibition effect of thenti-NSE biomacromolecules for electron transfer. Subsequently,et further increased after BSA and NSE were successively adsorbednto the electrode surface (Fig. 2, curves f and g, respectively),hich is consistent with the fact that the hydrophobic layer of therotein insulates the conductive support and binds the interfaciallectron transfer.

.3. Cyclic voltammetric characterization of immunosensor

CVs of different modified electrodes were performed in.1 mol/L PBS (pH 7.4) from 0 to 0.8 V (vs. SCE) at a scan rate of00 mV/s, as shown in Fig. 3A. No obvious redox peak (Fig. 3A,urve a) was observed at the bare GCE as the lack of redox medi-tor. Due to the porous AuNCs film increased the surface area oflectrode, it can be observed an increase in the background cur-ent can be found after the modification of AuNCs film (Fig. 3A,

urve b). In order to further illuminate the difference of bareCE and AuNCs/GCE, CVs for the bare GCE and AuNCs/GCE in.1 mol/L PBS (pH 7.4) and 5.0 mmol/L Fe(CN)6

3−/4− are also shownn Fig. 3A (insert of the top left corner and the below right corner,

ctronics 31 (2012) 399– 405

respectively). After NiHCFNPs were immobilized on the AuNCs/GCEand the resulting electrode showed a pair of typical reversible redoxpeak (Fig. 3A, curve c), indicating NiHCFNPs is an excellent elec-troactive substance and favorably enhance transfer of the electron.Compared with curve c, the peak current (Fig. 3A, curve d) fur-ther increased after Au–Gra loading, which can be attributed to thegood conductivity of Au–Gra. With the immobilization of anti-NSEon the modified electrode surface, the peak current was decreased(Fig. 3A, curve e). And then blocking with 0.25% BSA, the peakcurrent was further decreased (Fig. 3A, curve f). The reason maybe that biological macromolecular protein BSA could hinder thetransmission of electrons on the electrode surface. Subsequently,a dramatically decrease in current is found (Fig. 3A, curve g) afterthe immunosensor was incubated in 1 ng/mL of NSE for 30 min, dueto the formation of immunocomplex which blocking the tunnel formass and electron transfer.

Moreover, we have studied electrochemical catalysis ofNiHCFNPs in the presence of DA. Fig. 3B shows CVs for the pro-posed immunosensor with and without DA. When DA was addedinto 0.1 mol/L PBS, the anodic peak current increased dramatically.Electrocatalytic oxidation mechanism of DA at the proposed elec-trode is shown in Scheme 1(B). On the basis of above results, wecan make a conclusion that the signal was amplified according toelectrocatalytic oxidation of DA by NiHCFNPs.

Furthermore, we also compared the detection efficiency of theimmunosensor with and without Au–Gra/NiHCFNPs nanomate-rials. Thus an anti-NSE/AuNCs modified GCE was prepared as acomparison immunosensor. Fig. 3C indicates that the peak currentincreased obviously on the anti-NSE/Au–Gra/NiHCFNPs/AuNCsmodified GCE, which further confirmed the role of Au–Gra andNiHCFNPs in improving the immunosensor sensitivity.

Supplementary Fig. 1 depicts the CVs of the preparedimmunosensor in 0.1 mol/L PBS (pH 7.4) from 0 to 0.8 V (vs. SCE) atdifferent scan rates. It is evident that both the anodic and anodicpeak currents are increased as the scan rates in the range from 20 to600 mV/s. In addition, the peak current versus the square of root ofsweep rate plot is shown in inset, which depicts a linear relationshipand suggests the reaction is a diffusion-controlled process.

3.4. Optimization of immunoassay conditions

3.4.1. Effect of pH on the response of the immunosensorAs the pH of the working buffer has a profound effect on the

amperometric responses, the pH dependence of the voltammetricresponse was investigated over the pH values from 4.0 to 9.0. Theresults indicated that the most strong peak current response wasobtained at pH 7.4. Thus, the pH 7.4 of PBS was used as the optimumbuffer solution in the further study.

3.4.2. Effect of temperature on the immunoreactionsThe effect of temperature on the performance of the

immunosensor was also examined from 18 to 45 ◦C. Obviously, anincrease of temperature had a favorable effect on the immunoreac-tion by constant concentrations of NSE before 37 ◦C. Actually, 37 ◦Cis the optimal temperature of immunoreaction. However, consid-ering the denaturation of protein at high temperature, incubationtemperature of 25 ◦C was adopted as the optimum immunoreactiontemperature.

3.4.3. Effect of incubation time on the immunoreactionsThe immunochemical incubation time was also studied. The

immunosensor was immersed in 1 ng/mL NSE for 10, 15, 20, 25,30, 35 and 40 min, respectively. It was found that the currentresponse decreased with the incubation time rapidly up to 30 minand then tended to level off, indicating the saturated formation of

J. Han et al. / Biosensors and Bioelectronics 31 (2012) 399– 405 403

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withou tAu-Gra/NiHCFNPs

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E (V)

Fig. 3. (A) CVs for (a) bare GCE; (b) AuNCs; (c) NiHCFNPs/AuNCs; (d) Au–Gra/NiHCFNPs/AuNCs; (e) anti-NSE/Au–Gra/NiHCFNPs/AuNCs modified GCE; and (f) anti-NSE/Au–Gra/NiHCFNPs/AuNCs modified GCE blocked with 0.25% BSA; (g) the proposed immunosensor after incubation with 1 ng/mL NSE in 0.1 mol/L PBS. The insertsshow the CVs for the bare GCE and AuNCs/GCE in 0.1 mol/L PBS (the top left corner) and in 5.0 mmol/L Fe(CN)6

3−/4− (the below right corner). (B) CVs for the proposedi the pw re giv

i3

3

tW

F0

mmunosensor with and without DA when incubation with 1 ng/mL NSE. (C) CVs forhen incubation with 1 ng/mL NSE. The scan rate was 100 mV/s and all potentials a

mmunocomplex on the modified electrode. Therefore, we chose0 min as the optimal incubation time for the subsequent work.

.4.4. Influence of concentration of DAThe influence of concentration of DA in 0.1 mol/L PBS for elec-

rochemical catalysis of NiHCFNPs was studied using CVs (Fig. 4).ith increasing the concentration of DA, the oxidation peak current

0

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C DA (μmol/L)10 20 30 5040

I (μ

A)

35

ig. 4. The current response with various concentration of DA when incubation with.001 ng/mL NSE.

roposed immunosensor with and without Au–Gra/NiHCFNPs in the presence of DAen versus SCE.

increased gradually, which indicates that DA could be electrocat-alytic oxidated by NiHCFNPs. The maximum peak current responseoccurred in 40 �mol/L, which corresponded to the saturated state.Consequently, the optimum concentration of 40 �mol/L DA wasemployed for the further study.

3.5. Performance of the immunosensor

3.5.1. CV response and calibration curveThe calibration plot for NSE detection with the proposed

immunosensor under optimal experimental conditions is illus-trated in Fig. 5A. As expected, the peak currents decrease with theincreasing concentration of NSE after antigen–antibody reaction.The reason might be that the formed immunocomplex could hin-der the electron transfer towards the electrode surface. The changeof the �I was proportional to the logarithm of NSE concentrationin the range of 0.001–100 ng/mL with a regression equation of �I(�A) = 226.28 + 60.84 log CNSE (ng/mL) and correlation coefficient of0.9924. The detection limit corresponding to three times the stan-dard deviation of the blank solution was estimated as 0.3 pg/mL(Fig. 5A, curve 1). As a comparative study, the current responsesof the proposed immunosensors were recorded without the addi-tion of DA, the linear range was from 0.005 to 100 ng/mL, the linearregression equation was �I (�A) = 104.96 + 43.79 log CNSE (ng/mL)

and the detection limit of 2.0 pg/mL (Fig. 5A, curve 2). These resultsreveal that DA could be electrocatalytic oxidated by NiHCFNPs. Atthe same time, CVs for the prepared immunosensors with the addi-tion of DA and without DA are shown in Fig. 5A and B, respectively.

404 J. Han et al. / Biosensors and Bioelectronics 31 (2012) 399– 405

210-1-2-30

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Fig. 5. (A) The comparison of calibration plots of the change of anodic peak current response versus the logarithm of concentrations of NSE with the different immunosensorsu works /s. (B)i repre

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ee2is

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3

ippmi

nder optimal conditions: (1) anti-NSE/Au–Gra/NiHCFNPs/AuNCs/GCE with DA inolution, respectively. All potentials are given vs. SCE and the scan rate was 100 mVmmunosensor anti-NSE/Au–Gra/NiHCFNPs/AuNCs/GCE without DA. The error bars

.5.2. Selectivity, reproducibility, stability of the immunosensorSelectivity is the important performance of immunosensors. To

ssess the selectivity of the proposed immunosensor, we stud-ed the two batches of modified electrodes with and withoutSE in the same conditions. The one batch of the proposed

mmunosensors was incubated with 1 ng/mL NSE, 50 ng/mL pro-astrin releasing-peptide (ProGRP), 50 ng/mL carcinoembryonicntigen (CEA), 50 ng/mL �-fetoprotein antigen (AFP) or 1 �g/mLSA, respectively. The charge of the �I is 218.7 �A, 14.24 �A,0.87 �A, 12.41 �A, 4.20 �A, respectively. The other batch of theroposed immunosensors was incubated with 1 ng/mL NSE and

ng/mL NSE containing the same concentration of above inter-erences (ProGRP, CEA, AFP and BSA). The change of the currentesponses is 4.2%, 3.1%, 2.4% and 2.8%, respectively, which indi-ates that the selectivity of the immunosensor based on the specificntigen–antibody immunoreactions is acceptable.

The reproducibility of the response of the immunosensors wasvaluated by determining 1 ng/mL NSE using five equally proposedlectrodes. The immunosensors show the change of the �I of 231.2,13.9, 208.6, 217.2, 223.0 �A, respectively. It is observed that the

mmunosensors have acceptable reproducibility with a relativetandard deviation (RSD) of 4.0% (n = 5).

The stability of the BSA/Au–Gra/NiHCFNPs/AuNCs/GCE was alsoxamined. When the prepared immunosensor were not in use, theyere stored in PBS (pH 7.4) at 4 ◦C. After storing for 30 days, the

esponse changed less than 7.97% compared with the initial steadytate value. Therefore, the selectivity, reproducibility and stabilityf the immunosensor are acceptable.

.6. Clinical application

To further evaluate the applicability of the developedmmunoassay method, six serum samples from the Ninth Peo-

le’s Hospital of Chongqing, China, were analyzed by theroposed immunosensor. The results were compared with com-ercially enzyme-linked immunosorbent assay (ELISA) as shown

n Supplementary Table 1. The relative deviation of these results

ing solution; (2) anti-NSE/Au–Gra/NiHCFNPs/AuNCs/GCE without DA in working CVs of immunosensor anti-NSE/Au–Gra/NiHCFNPs/AuNCs/GCE with DA. (C) CVs ofsent the standard deviations of three parallel samples at each target concentration.

is between −7.06% and 7.42%, which revealed that the proposedimmunosensor is in good agreement with ELISA.

4. Conclusions

In summary, we described an ultrasensitive signal ampli-fied strategy by electrochemical catalysis DA for label-free tracetumor marker detection. The greatly enhanced sensitivity relieson multiple signal amplification: firstly, we take advantage ofdual-effects Au–Gra for enhancing the electroactivity of NiHCFNPslayer and increasing the amount of anti-NSE loading. Secondly,NiHCFNPs are one of the excellent electroactive nanomaterialsand favorably enhance the oxidation peak current by the electro-chemical catalysis of DA, resulting in the further improvement ofthe immunosensor sensitivity. Thus, the proposed immunosensorshows a lower detection limit and wider linear range, higher sensi-tivity, and provides a promising signal amplified strategy for clinicalimmunoassay.

Acknowledgements

The authors are grateful for the financial support by theNNSF of China (21105081, 21075100), Ministry of Educa-tion of China (Project 708073), Specialized Research Fund forthe Doctoral Program of Higher Education (20100182110015),State Key Laboratory of Electroanalytical Chemistry (SKLEAC2010009) and Natural Science Foundation Project of ChongqingCity (CSTC-2010BB4121, CSTC-2011BA7003, CSTC-2009BA1003),the Fundamental Research Funds for the Central Universi-ties (XDJK2010C062, XDJK2009B013), the Doctor Foundation ofSouthwest University (SWU109016) and the Outstanding YouthFoundation of College of Chemistry and Chemical Engineering,Southwest University (SWUC009), China.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bios.2011.10.055.

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