韩静-BB

韩静-BB
韩静-BB

Biosensors and Bioelectronics 31 (2012) 399–405

Contents lists available at SciVerse ScienceDirect

Biosensors and

Bioelectronics

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /b i o

s

Novel electrochemical catalysis as signal ampli?ed strategy for label-free detection of neuron-speci?c enolase

Jing Han,Ying Zhuo ?,Ya-Qin Chai,Ya-Li Yuan,Ruo Yuan ?

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

a r t i c l e

i n f o

Article history:

Received 29July 2011

Received in revised form 25October 2011Accepted 27October 2011

Available online 25 November 2011

Keywords:

Immunosensor

Electrochemical catalysis

Gold nanoparticles functionalized graphene nanosheets (Au–Gra)

Nickel hexacyanoferrates nanoparticles (NiHCFNPs)Dopamine (DA)

Neuron-speci?c enolase (NSE)

a b s t r a c t

A label-free electrochemical immunoassay for neuron-speci?c enolase (NSE),a kind of lung cancer marker,was developed in this work via novel electrochemical catalysis for signal ampli?cation.The new ampli?ed 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 gold nanocrystals (AuNCs)modi?ed glassy carbon electrode (GCE),could exhibit a distinct pair of redox peaks corresponding 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 ?lm.Then an enhanced amount of neuron-speci?c 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 and large speci?c 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 the further improvement of the immunosensor sensitivity.Under optimal conditions,the electrochemical immunosensor exhibited a linear range of 0.001–100ng/mL with a detection limit of 0.3pg/mL (S/N =3).Thus,the proposed immunosensor provides a rapid,simple,and sensitive immunoassay protocol for NSE detection,which may hold a promise for clinical diagnosis.

? 2011 Elsevier B.V. All rights reserved.

1.Introduction

Nowadays,cancer is one of the most threatening diseases for human beings.The quantitative detection of tumor biomarkers,including serum tumor markers and potential prognostic factors,plays an important role in clinical early diagnosis (Faraggi and Kramar,2000).Neuron-speci?c enolase (NSE)is a sensitive,speci?c,and reliable tumor markers for small-cell lung carcinoma (SCLC)at the time of diagnosis (Oremek et al.,2007;Erbaycu et al.,2010).As the ?-subunit of enolase,NSE presents primarily in the cytoplasm of neurons and neuroendocrine cells.The NSE levels are 5–12ng/mL in serum and 20ng/mL in cerebrospinal ?uid in normal human beings (Marangos and Schmechel,1987).Therefore,the determination of NSE level is of great importance to process the early diagnostic and prognostic values for monitoring the SCLC state.

Electrochemical immunosensors,based on the speci?c antigen–antibody recognition,have gained wide interest in detection and quanti?cation of bio-molecules (Wu et al.,2007;Arruda et al.,2009).Recently,increasing interests have been focused on label-free amperometric immunosensor due to their

?Corresponding authors.Tel.:+862368252277;fax:+862368252277.E-mail addresses:yingzhuo@https://www.360docs.net/doc/c31444526.html, ,yingzhuo@https://www.360docs.net/doc/c31444526.html, (Y.Zhuo),yuanruo@https://www.360docs.net/doc/c31444526.html, (R.Yuan).

rapid recognition,simple fabrication and operation (Okuno et al.,2007;Mun et al.,2010).However,the sensitivity has been limited as the lack of signal ampli?ed strategy for most label-free amper-ometric https://www.360docs.net/doc/c31444526.html,tely,there are some reports of the label-free amperometric immunosensors based on enzyme biocat-alytic ampli?ed strategy (Zhuo et al.,2006;Liu et al.,2011),but the defect of irreversible enzyme inactivation and relatively expen-sive of enzyme protein restricts the further application.Nickel hexacyanoferrate nanoparticles (NiHCFNPs)have been reported to be a versatile nanomaterial for the label-free immunosensor construction because of their well-de?ned and reproducible redox voltammetric responses.Furthermore,as a kind of small biomolecule with redox activity,it was reported by Prabhu et al.(2011)dopamine (DA)could be electrocatalytic oxidated by metal hexacyanoferrate.Thus,a new ampli?ed strategy based on the electrochemical catalysis of NiHCFNPs in the presence of DA was developed for immunosensor construction in this work.

Biomolecular immobilization has been considered as one of the most important points in biosensor fabrication.Au nanoparticles have been recognized as the ideal nanomaterials for biomolecular immobilization as they could adsorb many biomolecules and main-tain their bioactivities (Ding et al.,2004;Wei et al.,2007;Shan et al.,2010).Recently,an explosion of interest has been focused on graphene (Gra)which possesses many extraordinary proper-ties (Kaner,2008;Geim,2009;Wang et al.,2010),including good

0956-5663/$–see front matter ? 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.bios.2011.10.055

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

405

Scheme 1.Illustration of the stepwise immunosensor fabrication process:(a)formation of AuNCs ?lm;(b)assembled the NiHCFNPs;(c)formation of Au–Gra monolayer;(d)anti-NSE loading;(e)blocking with 0.25%BSA;(f)incubation with NSE.(A)TEM image of Au–Gra.(B)Electrocatalytic oxidation mechanism of DA at the prepared electrode.(C)CV measurements of current response with different concentrations of NSE in the presence of DA.

mechanical properties,large surface area,an excellent electron transfer rate for extensive applications in sensors,electrochemi-cal devices,polymer nanocomposites and so on (Ramanathan et al.,2008;Xia et al.,2010;Huang et al.,2011).Therefore,we take advan-tage of dual-effects of gold nanoparticles and graphene nanosheets to prepare the gold nanoparticles functionalized graphene sheets (Au–Gra)nanohybrid,which exhibited an enhanced adsorption for antibody immobilization (Han et al.,2011).

In this work,a novel electrocatalytic strategy was developed for the fabrication of a label-free NSE immunosensor.The large surface area of Au–Gra can increase the amount of anti-NSE loading and the good conductivity of Au–Gra can also enhance the electroactivity of NiHCFNPs.NiHCFNPs are one of the excellent electroactive nano-materials and favorably enhance the oxidation peak current by the electrochemical catalysis of DA.Experiment results show that the amperometric response of the immunosensor could be ampli?ed which can enormously improve the sensitivity of immunosensor.The details of the attractive response performances of the pro-posed immunosensor and potential merits for NSE detection are substantiated as follows.

2.Experimental

2.1.Reagent and materials

NSE (0–100ng/mL)and NSE monoclonal antibody (anti-NSE)were purchased from Advanced Life Science Institute,Inc.,Saitama,Japan.Graphene oxide sheets (GO)were obtained in Pioneer Nanotechnology Co.(Nanjing,China).DA was purchased from Chemical Reagent Co.(Chongqing,China).Bovine serum albu-min (BSA,96–99%),gold chloride (HAuCl 4),sodium citrate and l -ascorbic acid (AA)were obtained from Sigma Chemical Co.(St.Louis,MO,USA).K 3Fe(CN)6and NiCl 2·6H 2O were obtained from Chemical Reagent Co.(Sichuan,China).All other materi-als used were of the highest quality available and purchased from regular sources.Bi-distilled water was used throughout this study.Phosphate buffered solutions (PBS)(pH 7.4)were pre-pared using 0.1mol/L Na 2HPO 4,0.1mol/L KH 2PO 4,and 0.1mol/L NaCl.

2.2.Apparatus

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

Hua Instrument,Co.,China).The AC impedance of the immuno-electrode membrane was measured with a Model IM6e (ZAHNER Elektrick,Germany).A three-electrode electrochemical system was composed of a platinum wire as the auxiliary electrode,a saturated calomel electrode (SCE)as the reference electrode and a modi?ed glassy carbon electrode (GCE,?=4mm)as the working electrode.The topograghs of the Au substrates mod-i?ed with different materials were investigated with atomic force microscopy (AFM,Veeco,Woodbury,NY,USA).The size of gold nanoparticles functionalized graphene nanosheets was estimated from transmission electron microscopy (TEM)(H600,Hitachi Instrument,Japan).The pH measurements were made with a 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).Brie?y,40mL of 0.01mol/L NiCl 2aqueous solution was ?rst dropped gradually into 40mL of 0.05mol/L K 3Fe(CN)6solution containing 0.05mol/L KCl under stir-ring.Then the resulting mixture solutions were vigorously agitated for 5min,and immediately centrifuged,washed with double dis-tilled water for three times.Subsequently,the NiHCFNPs were dried 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 ?rst,GO were dis-solved in water by ultrasonic dispersion.And then 0.1g AA was added to 10mL of an aqueous dispersion of GO (1mg/mL)and stirred for overnight at room temperature.Next,2mL of 1%gold chloride solution was added to the above mixture and stirred for 8h.Following that,in order to remove the excessive graphite oxide and AA,the product of Au–Gra were centrifugally washed extensively with double distilled water and ?nally dispersed in bi-distilled water.Herein,the Au–Gra were characterized by TEM,which indi-cated the gold nanoparticles were integrated uniformly with the graphene nanosheets by one step chemical method of reducing GO and HAuCl 4via AA under gentle conditions.And TEM of Au–Gra is presented in Scheme 1(A).

J.Han et al./Biosensors and Bioelectronics31 (2012) 399–405401

2.5.Fabrication of proposed immunosensor

To obtain mirror-like surface,the GCE(?=4mm)was?rstly polished successively with0.3and0.05?m alumina powder.And then it was rinsed with distilled water and ethanol in ultrasonic bath to remove the physically absorbed substance.After that,the GCE was allowed to dry at room temperature.

Firstly,the pretreated GCE was immersed in2mL of1%HAuCl4 solution for electrochemical deposition under constant potential of?0.2V for30s to obtain porous gold nanocrystals(AuNCs)?lm modi?ed electrode(AuNCs/GCE).Then,10?L prepared NiHCFNPs aqueous solution was dropped on the surface of AuNCs layer as redox probe via the interaction of–CN groups of NiHCFNPs and AuNCs.Next,10?L Au–Gra was coated on NiHCFNPs membrane modi?ed electrode.Following that,the obtained electrode was incubated in0.5mL anti-NSE solution at4?C for12h.To block the possible remaining active sites and avoid the non-speci?c adsorp-tion,20?L of0.25%BSA solution was dropped on the electrode for 0.5h at37?C.After every step,the modi?ed electrode was thor-oughly cleaned with bi-distilled water to remove the physically absorbed species.The?nished immunosensor was stored at4?C when not in use.Scheme1may illustrate self-assembly proce-dure and the mechanism of the proposed electrochemical catalysis strategy.

2.6.Experimental measurements

The electrochemical properties of the modi?ed electrode were characterized by CV and EIS.The CV scan was taken from0to 0.8V at100mV/s in PBS(PH7.4).EIS measurements were done in the presence of a5.0mM K3[Fe(CN)6]/K4[Fe(CN)6](1:1)mixture as a redox probe at the formal potential of220mV.The alterna-tive voltage is10mV and the frequency range is from1×10?2 to1×106Hz.The detection is based on the change of the anodic peak current( I)response before and after the antigen–antibody reaction,due to the immunocomplex hindering the access of redox probe to electrode.When the background current was stabilized, the anodic peak current response was recorded as I0.Then,the immunoreaction was carried out,and after that the anodic peak current response was recorded as I.The change of the I was given by I=I0?I.

3.Results and discussion

3.1.Characteristics of the immunosensor

The surface topographic feature of the self-assemble proce-dure of the immunosensor was investigated via AFM.The images of different modi?ed gold surfaces are shown in Fig.1.Fig.1A indicates an image of the AuNCs?lm by electrodeposition,which displays a quite uniform triangle-like structure.Fig.1B exhibits a topograph with globular features ascribed to the uniform dis-tribution of NiHCFNPs in AuNCs?lm by covalent bonds and electrostatic interaction.After Au–Gra were assembled onto the resulting NiHCFNPs/AuNCs?lm,many nano-Au particles wrapped with a layer of thin?lm of Gra with the typical crumpled and wrinkled structure was obtained(Fig.1C).Such a hybrid mono-layer could present a large speci?c surface area,high surface free energy,good conductivity and a biocompatible environment. Subsequently,the anti-NSE adsorbed on Au–Gra/NiHCFNPs/AuNCs surface was observed from Fig.1D.The morphology exhibited an image of a dense protein layer and a compact sheet-shaped struc-ture due to the presence of Au–Gra,indicating that the anti-NSE was successfully absorbed onto the

electrode.Fig. 1.AFM images of differently modi?ed Au substrates:(a)AuNCs;

(b)NiHCFNPs/AuNCs;(c)Au–Gra/NiHCFNPs/AuNCs;(d)anti-NSE/Au–Gra/ NiHCFNPs/AuNCs.

402J.Han et al./Biosensors and Bioelectronics 31 (2012) 399–405

Z i m (Ω)

Z re ( Ω)

Fig. 2.EIS of different electrodes in 5.0mmol/L K 3[Fe(CN)6]/K 4[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-NSE/Au–Gra/NiHCFNPs/AuNCs;(g)NSE/BSA/anti-NSE/Au–Gra/NiHCFNPs/AuNCs.The frequency range is at 1×10?2to 1×106Hz at 25?C (Z im vs.Z re at 220mV vs.SCE).

3.2.Electrochemical impedance spectroscopy (EIS)of the modifying process

EIS is an effective method for studying the interface properties of surface-modi?ed electrodes and the electron-transfer resistance at the electrode surface.It is well known that the semicircle diameter of EIS is equal to R et in the Nyquist diagram.And the Nyquist dia-grams at different modi?ed electrodes in 5.0mmol/L Fe(CN)63?/4?solution are presented in Fig.2.The bare GCE exhibits a small semi-circle in the high frequency section (curve a).When the electrode deposited with HAuCl 4,the Nyquist diagram is approximately a straight line (curve b),indicating porous AuNCs ?lm has excel-lent conductive properties,which formed a network like structure and constructed larger effective surface area than bare GCE surface.Then NiHCFNPs were loaded on the AuNCs/GCE,the R et increased a litter but still less than the bare GCE (curve c).The reason may be that the microstructure of NiHCFNPs formed some barrier obstruct-ing the electron transfer.Next,the R et decreased obviously when Au–Gra were assembled on the NiHCFNPs surface (curve d),due to the Au–Gra could provide a large surface area and act as a conduct-ing tunnel to promote the electron transfer.When anti-NSE was immobilized onto the modi?ed electrode,the R et increased dra-matically (curve e),which is ascribed to the inhibition effect of the anti-NSE biomacromolecules for electron transfer.Subsequently,R et further increased after BSA and NSE were successively adsorbed onto the electrode surface (Fig.2,curves f and g,respectively),which is consistent with the fact that the hydrophobic layer of the protein insulates the conductive support and binds the interfacial electron transfer.

3.3.Cyclic voltammetric characterization of immunosensor

CVs of different modi?ed electrodes were performed in 0.1mol/L PBS (pH 7.4)from 0to 0.8V (vs .SCE)at a scan rate of 100mV/s,as shown in Fig.3A.No obvious redox peak (Fig.3A,curve a)was observed at the bare GCE as the lack of redox medi-ator.Due to the porous AuNCs ?lm increased the surface area of electrode,it can be observed an increase in the background cur-rent can be found after the modi?cation of AuNCs ?lm (Fig.3A,curve b).In order to further illuminate the difference of bare GCE and AuNCs/GCE,CVs for the bare GCE and AuNCs/GCE in 0.1mol/L PBS (pH 7.4)and 5.0mmol/L Fe(CN)63?/4?are also shown in Fig.3A (insert of the top left corner and the below right corner,

respectively).After NiHCFNPs were immobilized on the AuNCs/GCE and the resulting electrode showed a pair of typical reversible redox peak (Fig.3A,curve c),indicating NiHCFNPs is an excellent elec-troactive substance and favorably enhance transfer of the https://www.360docs.net/doc/c31444526.html,pared with curve c,the peak current (Fig.3A,curve d)fur-ther increased after Au–Gra loading,which can be attributed to the good conductivity of Au–Gra.With the immobilization of anti-NSE on the modi?ed electrode surface,the peak current was decreased (Fig.3A,curve e).And then blocking with 0.25%BSA,the peak current was further decreased (Fig.3A,curve f).The reason may be that biological macromolecular protein BSA could hinder the transmission of electrons on the electrode surface.Subsequently,a dramatically decrease in current is found (Fig.3A,curve g)after the immunosensor was incubated in 1ng/mL of NSE for 30min,due to the formation of immunocomplex which blocking the tunnel for mass and electron transfer.

Moreover,we have studied electrochemical catalysis of NiHCFNPs in the presence of DA.Fig.3B shows CVs for the pro-posed immunosensor with and without DA.When DA was added into 0.1mol/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,we can make a conclusion that the signal was ampli?ed according to electrocatalytic oxidation of DA by NiHCFNPs.

Furthermore,we also compared the detection ef?ciency of the immunosensor with and without Au–Gra/NiHCFNPs nanomate-rials.Thus an anti-NSE/AuNCs modi?ed GCE was prepared as a comparison immunosensor.Fig.3C indicates that the peak current increased obviously on the anti-NSE/Au–Gra/NiHCFNPs/AuNCs modi?ed GCE,which further con?rmed the role of Au–Gra and NiHCFNPs in improving the immunosensor sensitivity.

Supplementary Fig.1depicts the CVs of the prepared immunosensor in 0.1mol/L PBS (pH 7.4)from 0to 0.8V (vs .SCE)at different scan rates.It is evident that both the anodic and anodic peak currents are increased as the scan rates in the range from 20to 600mV/s.In addition,the peak current versus the square of root of sweep rate plot is shown in inset,which depicts a linear relationship and 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 immunosensor

As the pH of the working buffer has a profound effect on the amperometric responses,the pH dependence of the voltammetric response was investigated over the pH values from 4.0to 9.0.The results indicated that the most strong peak current response was obtained at pH 7.4.Thus,the pH 7.4of PBS was used as the optimum buffer solution in the further study.

3.4.2.Effect of temperature on the immunoreactions

The effect of temperature on the performance of the immunosensor was also examined from 18to 45?C.Obviously,an increase of temperature had a favorable effect on the immunoreac-tion by constant concentrations of NSE before 37?C.Actually,37?C is the optimal temperature of immunoreaction.However,consid-ering the denaturation of protein at high temperature,incubation temperature of 25?C was adopted as the optimum immunoreaction temperature.

3.4.3.Effect of incubation time on the immunoreactions

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

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

403

0.8

0.60.4

0.2

0.0

I (μA )

E (V)

0.8

0.6

0.4

0.2

0.0

I (μA )

E (V)

-20

I

(μA )

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 modi?ed GCE;and (f)anti-NSE/Au–Gra/NiHCFNPs/AuNCs modi?ed GCE blocked with 0.25%BSA;(g)the proposed immunosensor after incubation with 1ng/mL NSE in 0.1mol/L PBS.The inserts

show the CVs for the bare GCE and AuNCs/GCE in 0.1mol/L PBS (the top left corner)and in 5.0mmol/L Fe(CN)63?/4?(the below right corner).(B)CVs for the proposed immunosensor with and without DA when incubation with 1ng/mL NSE.(C)CVs for the proposed immunosensor with and without Au–Gra/NiHCFNPs in the presence of DA when incubation with 1ng/mL NSE.The scan rate was 100mV/s and all potentials are given versus SCE.

immunocomplex on the modi?ed electrode.Therefore,we chose 30min as the optimal incubation time for the subsequent work.

3.4.4.In?uence of concentration of DA

The in?uence of concentration of DA in 0.1mol/L PBS for elec-trochemical catalysis of NiHCFNPs was studied using CVs (Fig.4).With increasing the concentration of DA,the oxidation peak current

200

400

600

C DA (μmol/L)

I (μA )

Fig.4.The current response with various concentration of DA when incubation with 0.001ng/mL NSE.

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

3.5.Performance of the immunosensor

3.5.1.CV response and calibration curve

The 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 the increasing 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 change of the I was proportional to the logarithm of NSE concentration in the range of 0.001–100ng/mL with a regression equation of I (?A)=226.28+60.84log C NSE (ng/mL)and correlation coef?cient of 0.9924.The detection limit corresponding to three times the stan-dard deviation of the blank solution was estimated as 0.3pg/mL (Fig.5A,curve 1).As a comparative study,the current responses of the proposed immunosensors were recorded without the addi-tion of DA,the linear range was from 0.005to 100ng/mL,the linear regression equation was I (?A)=104.96+43.79log C NSE (ng/mL)and the detection limit of 2.0pg/mL (Fig.5A,curve 2).These results reveal that DA could be electrocatalytic oxidated by NiHCFNPs.At the same time,CVs for the prepared immunosensors with the addi-tion of DA and without DA are shown in Fig.5A and B,respectively.

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

100

200

300

(A)

ΔI (μA )

logC NSE

(ng/mL)

(B)

I (μA )

E (V)

I (μA )

E (V)

(C)

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 immunosensors

under optimal conditions:(1)anti-NSE/Au–Gra/NiHCFNPs/AuNCs/GCE with DA in working solution;(2)anti-NSE/Au–Gra/NiHCFNPs/AuNCs/GCE without DA in working solution,respectively.All potentials are given vs.SCE and the scan rate was 100mV/s.(B)CVs of immunosensor anti-NSE/Au–Gra/NiHCFNPs/AuNCs/GCE with DA.(C)CVs of immunosensor anti-NSE/Au–Gra/NiHCFNPs/AuNCs/GCE without DA.The error bars represent the standard deviations of three parallel samples at each target concentration.

3.5.2.Selectivity,reproducibility,stability of the immunosensor

Selectivity is the important performance of immunosensors.To assess the selectivity of the proposed immunosensor,we stud-ied the two batches of modi?ed electrodes with and without NSE in the same conditions.The one batch of the proposed immunosensors was incubated with 1ng/mL NSE,50ng/mL pro-gastrin releasing-peptide (ProGRP),50ng/mL carcinoembryonic antigen (CEA),50ng/mL ?-fetoprotein antigen (AFP)or 1?g/mL BSA,respectively.The charge of the I is 218.7?A,14.24?A,10.87?A,12.41?A,4.20?A,respectively.The other batch of the proposed immunosensors was incubated with 1ng/mL NSE and 1ng/mL NSE containing the same concentration of above inter-ferences (ProGRP,CEA,AFP and BSA).The change of the current responses is 4.2%,3.1%,2.4%and 2.8%,respectively,which indi-cates that the selectivity of the immunosensor based on the speci?c antigen–antibody immunoreactions is acceptable.

The reproducibility of the response of the immunosensors was evaluated by determining 1ng/mL NSE using ?ve equally proposed electrodes.The immunosensors show the change of the I of 231.2,213.9,208.6,217.2,223.0?A,respectively.It is observed that the immunosensors have acceptable reproducibility with a relative standard deviation (RSD)of 4.0%(n =5).

The stability of the BSA/Au–Gra/NiHCFNPs/AuNCs/GCE was also examined.When the prepared immunosensor were not in use,they were stored in PBS (pH 7.4)at 4?C.After storing for 30days,the response changed less than 7.97%compared with the initial steady state value.Therefore,the selectivity,reproducibility and stability of the immunosensor are acceptable.

3.6.Clinical application

To further evaluate the applicability of the developed immunoassay method,six serum samples from the Ninth Peo-ple’s Hospital of Chongqing,China,were analyzed by the proposed immunosensor.The results were compared with com-mercially enzyme-linked immunosorbent assay (ELISA)as shown in Supplementary Table 1.The relative deviation of these results

is between ?7.06%and 7.42%,which revealed that the proposed immunosensor is in good agreement with ELISA.

4.Conclusions

In summary,we described an ultrasensitive signal ampli-?ed strategy by electrochemical catalysis DA for label-free trace tumor marker detection.The greatly enhanced sensitivity relies on multiple signal ampli?cation:?rstly,we take advantage of dual-effects Au–Gra for enhancing the electroactivity of NiHCFNPs layer and increasing the amount of anti-NSE loading.Secondly,NiHCFNPs are one of the excellent electroactive nanomaterials and favorably enhance the oxidation peak current by the electro-chemical catalysis of DA,resulting in the further improvement of the immunosensor sensitivity.Thus,the proposed immunosensor shows a lower detection limit and wider linear range,higher sensi-tivity,and provides a promising signal ampli?ed strategy for clinical immunoassay.

Acknowledgements

The authors are grateful for the ?nancial support by the NNSF of China (21105081,21075100),Ministry of Educa-tion of China (Project 708073),Specialized Research Fund for the Doctoral Program of Higher Education (20100182110015),State Key Laboratory of Electroanalytical Chemistry (SKLEAC 2010009)and Natural Science Foundation Project of Chongqing City (CSTC-2010BB4121,CSTC-2011BA7003,CSTC-2009BA1003),the Fundamental Research Funds for the Central Universi-ties (XDJK2010C062,XDJK2009B013),the Doctor Foundation of Southwest University (SWU109016)and the Outstanding Youth Foundation of College of Chemistry and Chemical Engineering,Southwest University (SWUC009),China.

Appendix A.Supplementary data

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

J.Han et al./Biosensors and Bioelectronics31 (2012) 399–405405

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