journal of colloid and interface science

Amperometric immunosensor for the determination of a -1-fetoprotein based on multiwalled carbon nanotube–silver nanoparticle composite

Xin Che,Ruo Yuan *,Yaqin Chai,Jingjing Li,Zhongju Song,Jinfen Wang

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

a r t i c l e i n f o Article history:

Received 10October 2009Accepted 13January 2010

Available online 6February 2010Keywords:

Amperometric immunosensor

Multiwalled carbon nanotube–silver composite MnO 2

a -1-Fetoprotein

a b s t r a c t

A new amperometric immunosensor for the determination of a -1-fetoprotein (AFP)has been con-structed.First,a multiwalled carbon nanotube–silver (MWNT–Ag)composite was modi?ed on the sur-face of a glassy carbon electrode.Then,chitosan–MnO 2(CS–MnO 2)with excellent ?lm forming ability was dipped onto the MWNT–Ag-modi?ed electrode.Subsequently,gold nanoparticles were electrode-posited on the electrode to immobilize anti-AFP.The assembly processes were characterized with cyclic voltammetry,electrochemical impedance spectroscopy,and scanning electron microscopy.MWNT–Ag composite was characterized by transmission electron microscopy,X-ray photoelectron spectroscopy,and UV–vis spectroscopy.The system was optimized to realize a reliable determination of AFP in the range of 0.25–250ng/ml with a detection limit of 0.08ng/ml (S /N =3).The proposed immunosensor showed a rapid and highly sensitive amperometric response to AFP with acceptable stability and reproducibility.

ó2010Elsevier Inc.All rights reserved.

1.Introduction

a -1-Fetoprotein (AFP),an oncofetal glycoprotein,can be widely

used as a tumor maker found in several malignant diseases includ-ing hepatocellular cancer,yolk sac cancer,testicular cancer,and some other noncancerous diseases [1,2].In some cases,the level of AFP is related to the stage of the disease.An increased AFP con-centration in adult plasma may be an early indication of some can-cerous diseases [3,4].Therefore,the detection of AFP is necessary for clinical diagnosis.Among various immunoassay methods [5,6],electrochemical immunosensors have become a powerful method for detecting AFP because of their convenience,simple preparation,fast analytical time,low detection limit,etc.[7,8].Furthermore,with the development of nanotechnology,the use of nanoparticles to fabricate amperometric immunosensors has attracted widespread attention,due to their biocompatibility and high surface-to-volume ratio [9].But aggregation of nanoparticles prohibits extensive applications;thus,highly dispersed metal nano-particles are very important in fabricating an immunosensor [10].In order to resolve this problem,some successful methods have been reported to stabilize metal nanoparticles such as capping by poly-electrolytes or stabilizing in silicate-based matrix [11,12].Recently,there has been increasing interest in multiwalled carbon nanotubes (MWNTs)as heterogeneous catalyst supports.MWNTs possess unique properties such as high chemical stability,good electrical conductivity,tubular structure,high surface-to-volume ratio,and

strong adsorptive ability [13,14].The high surface area of MWNTs provides a large loading capacity for the nanoparticles.Studies have shown that metal nanoparticles supported on MWNTs may provide much improved catalytic activity [15,16].Gold [17],silver [14,18],copper [19],and platinum [20,21]have been modi?ed onto MWNTs successfully.However,silver nanoparticles have attracted consider-able interest because they not only have common characteristics of noble metal nanoparticles but also have unique properties of high catalytic activity and excellent surface-enhanced Raman scattering (SERS)[10,18].In this work,we used MWNTs as a template to syn-thesize MWNT–Ag composites.

However,MWNT–Ag composites can possibly leak from the electrode.Therefore,chitosan–MnO 2(CS–MnO 2)has been success-fully introduced.CS is a natural cationic polymer with abundant amino and hydroxy groups,and can prepare a robust ?lm on ac-count of its good biocompatibility,low toxicity,and excellent ?lm forming ability [22].MnO 2has particular physical and chemical properties and is wildly applied in catalysis and electrodes in lith-ium https://www.360docs.net/doc/c03206821.html,pared with other transition metal oxides,MnO 2is more attractive in terms of its low cost,abundance,and small harmful nature [23,24].Besides,their performances are interre-lated.CS provides a large network structure for dispersing MnO 2uniformity,and MnO 2changes the friability of CS and improves the stability during redox cycling [25].

In order to adsorb biomolecules and maintain their bioactivity,gold nanoparticles (nano-Au)are introduced on account of their large speci?c surface area,strong adsorption ability,and good conductivity [7,26].Up to now,various methods for the fabrication of nano-Au have been reported,including chemical reduction,

0021-9797/$-see front matter ó2010Elsevier Inc.All rights reserved.doi:10.1016/j.jcis.2010.01.033

*Corresponding author.Fax:+862368253172.E-mail address:yuanruo@https://www.360docs.net/doc/c03206821.html, (R.Yuan).

Journal of Colloid and Interface Science 345(2010)

174–180

Contents lists available at ScienceDirect

Journal of Colloid and Interface Science

https://www.360docs.net/doc/c03206821.html, /l o c a t e /j c i

s

photolysis,radiolysis,and two-phase synthesis methods.However, those methods are time-consuming and the stability of as-pre-pared colloids is poor[11].We prepare nano-Au through an elec-trodeposition method which is convenient and rapid on CS–MnO2/MWNT–Ag?lm.

Taking into account the above remarks,this study developed a new strategy to construct AFP immunosensors by combining the merits of MWNT–Ag,CS–MnO2,and nano-Au.The ef?ciency of immunosensors can be enhanced because of the synergistic action between Ag nanoparticles and MnO2[27].The optimization of test conditions and the performances of the resulting immunosensor are reported in the experimental section.Moreover,the prepared immunosensor exhibited high sensitivity,low detection limit,and good stability.

2.Experimental

2.1.Reagents and chemicals

Anti-AFP and AFP were purchased from Biocell Company (Zhengzhou,China).Multiwalled carbon nanotubes(>95%purity) were purchased from Chengdu Organic Chemicals Co.Ltd.of the Chinese Academy of Science.Bovine serum albumin(BSA,96–99%),gold chloride(HAuCl4),chitosan(CS,deacetylating grade: 70–85%),and polyethyleneimine(PEI)were the products of Sigma Chemical Co.(St.Louis,MO,USA).All the other chemicals em-ployed were analytical grade and were used as received.Phos-phate-buffered solutions(PBS)at various pH were prepared using 0.1M Na2HPO4,0.1M KH2PO4,and0.1M KCl.Double-distilled water was used throughout the experiments.AFP and anti-AFP were stored at4°C before use.Standard solutions of anti-AFP were freshly prepared with PBS as in use.

2.2.Apparatus

Cyclic voltammetric measurements were carried out with a CHI 660A electrochemistry workstation(Shanghai,China).The conven-tional three-electrode system included a modi?ed electrode as working electrode,a saturated calomel reference electrode(SCE), and a platinum wire auxiliary electrode.UV–vis absorption spectra used a UV–vis spectrometer(UV–vis8500).Electrochemical impedance spectroscopy(EIS)measurements were carried out with a Model IM6e(Zahner,Germany).Scanning electron micro-graphs were studied with a scanning electron microscope(SEM, S-4800,Hitachi,Japan).The structure of the MWNT–Ag was inves-tigated with transmission electron microscopy(TEM,Tecnai,Ho-land).An X-ray photoelectron spectroscopy(XPS)spectrometer (ESCALAB250,Thermoelectricity Instruments,USA)was used to analyze the component of the MWNT–Ag composite.

2.3.Synthesis of MWNT–Ag composites

MWNT–Ag composites were synthesized according to Ref.[28] with a little change.The synthesis process was described as fol-lows.First,5ml of4mM AgNO3solution was slowly added into 5ml of2mg/ml PEI solution at pH9.0–9.1under magnetic stirring to prepare PEI–Ag+mixture solutions.The solution was stirred for 1h for completion of the coordination reaction.Second,MWNTs (2mg)which were puri?ed by acid treatment were suspended in 20ml water by sonication to make MWNTs disperse uniformly. Subsequently,5ml of the above prepared PEI–Ag+solution was added to the MWNT solution,followed by a combination of vigor-ous stirring and sonication.After they were mixed completely, NaBH4was added quickly to the mixtures for the complete reduc-tion of silver.After5min,the mixture was centrifuged and washed with doubly distilled water several times.The resulting products were dispersed in5ml water.

2.4.Preparation of CS–MnO2suspension

MnO2nanoparticles were prepared according to the literature [29],dispersed in water(20mg/ml),and then stored at4°C before use.CS–MnO2suspension(3mg/L)was prepared by mixing MnO2 with0.25%CS.

2.5.Preparation of the immunosensor

Before the modi?cation,bare glassy carbon electrodes(GCE) were polished successively with alumina slurries(0.3and 0.05l m),thoroughly rinsed,and sonicated in ethanol and double-distilled water each for5min.Then,10l l of MWNT–Ag composite was dipped onto the electrode.When water evaporated,10l l of CS–MnO2suspension was dipped onto the MWNT–Ag-modi?ed electrode.After drying in air,the obtained electrode was soaked in 3ml HAuCl4(1%)and electrodeposited atà0.2V for20s.After wash-ing with distilled water,the electrode was immersed into anti-AFP solution(0.3mg/ml)for8h at4°C,and then incubated into BSA solution(0.25wt.%)for about1h at room temperature to block pos-sible remaining active sites.The obtained electrode was stored at 4°C when not in use.The schematic diagram of the stepwise proce-dure of the immunosensor is shown in Scheme1.

2.6.Experimental measurements

Electrochemical impedance spectroscopy measurements were carried out in the presence of5mM K3[Fe(CN)6]/K4[Fe(CN)6](1/ 1)mixture as a redox probe in0.1M PBS(pH7.0).The alternative voltage was5mV and the frequency range is50–10kHz.

The electrochemical characteristics of the modi?ed electrode were characterized by cyclic voltammetry(CV).Electrochemical measurements were done in a conventional electrochemical cell at room temperature.CV scans were taken fromà0.6to0.7V(ver-sus SCE)with a scan rate of100mV/s in5ml0.1M PBS(pH7.0). The amperometric detection is based on the change of the peak current response(D I)before and after antigen–antibody reaction.

D I is evaluated as in the equation D I=IàI0,where I0represents the current response before the immunoreaction,and I represents the current response after

immunoreaction.

Scheme1.Schematic illustration of stepwise fabrication process of the immunosensor.

X.Che et al./Journal of Colloid and Interface Science345(2010)174–180175

3.Results and discussion

3.1.Characterization of MWNT–Ag composite

UV–vis spectroscopy was used to investigate the composite.As shown in Fig.1a,MWNT–Ag composite exhibited a UV–vis absorp-tion peak centered at408nm,which was the characteristic absorp-tion of Ag NPs and indicated the formation of Ag NPs[30].However, no absorption peak was observed from pure MWNT solution (Fig.1b).The microstructure of MWNT–Ag composite was also ob-served by TEM.As seen from Fig.2A,Ag NPs were distributed on the MWNT surface uniformly and the diameter of the MWNT–Ag ex-panded compared with the puri?ed MWNTs(Fig.2B).It indicated that PEI–Ag composites have attached onto the surface of MWNTs.

In order to further con?rm the existence of both Ag NPs and MWNTs in the MWNT–Ag composite,XPS experiments were em-ployed to characterize the chemical composition in the composite (Fig.3).Fig.3A was the XPS spectrum of the MWNT–Ag composite in a wide scan.The binding energies of368.2and373.3eV shown in Fig.3B correspond to the peaks of Ag3d5/2and Ag3d3/2[14], respectively,indicating the existence of Ag NPs in the composite. The C1s peak was also detected at a binding energy of284.6eV [14],which was corresponding to the C component of MWNTs (Fig.3C).The XPS peak of the binding energy assigned to the N1s component of PEI was detected at399.5eV as shown in Fig.3D[28].

3.2.SEM characterization of different membranes

The morphologies and microstructures of the MWNT–Ag,CS–MnO2/MWNT–Ag,and nano-Au/CS–MnO2/MWNT–Ag membrane were characterized using SEM.We can see MWNT–Ag composites dispersed uniformly on the electrode surface(Fig.4A).When CS–MnO2was assembled on the MWNT–Ag?lm(Fig.4B),a compact and rough surface was observed,which was attributed to the good ?lm forming ability of CS–MnO2.MWNT–Ag composites cannot be observed because they were embedded in the CS–MnO2matrix. After nano-Au was deposited on the CS–MnO2/MWNT–Ag mem-brane,we can see many different diameter globular particles uni-formly distributed on the?lm;some gold nanoparticles aggregated into larger nanoarchitectures with morphology like pinecones which have large speci?c surface areas(Fig.4C).

3.3.Electrochemical behavior of the modi?ed electrode

EIS is an effective method for probing the interfacial properties of surface-modi?ed electrodes.In the EIS,the linear part represents the diffusion-limited process.The semicircle portion in EIS corre-sponds to the electron transfer-limited process.The electron trans-fer resistance(R et)at the electrode surface is equal to the semicircle diameter.This resistance controls the electron transfer kinetics of the redox probe at the electrode interface.Therefore, EIS was used to characterize the assembly process of the electrode in this paper(Fig.5A).Bare GCE electrode had a small semicircle (curve a),implying low R et to the redox probe dissolved in electro-lyte solution.After MWNT–Ag was modi?ed onto the electrode,the resistance decreased compared with bare electrode(curve b).The reasons were that MWNT–Ag-mod?ed electrodes had larger effec-tive surface areas than bare GCE and MWNT–Ag composites had good conductivity.Then an increase of the interfacial resistance was observed when CS–MnO2was immobilized on MWNT–Ag, which was ascribed to CS–MnO2membrane increasing the?lm thickness and hindering the transmission of electrons.After depos-ite in HAuCl4,the R et decreased(curve d)owing to nano-Au playing an important role similar to a conducting wire to promote electron transfer[31].However,when anti-AFP and BSA were immobilized onto CS–MnO2/MWNT–Ag?lm,the resistances for the modi?ed electrode increased gradually,which were caused by the noncon-ductive properties of anti-AFP and BSA which acted as an inert electron layer and retarded the electron transfer.After the pre-pared immunosensor was incubated with AFP antigen solution (curve g),a larger increase of the resistance was shown.

In order to further con?rm the assembly process of the elec-trode,the assembly process of the multilayer?lms was also mon-itored by CV experiments(Fig.5B).No electrochemical peaks were observed at bare electrode(curve a).After MWNT–Ag was coated on the GCE surface,the resulting electrode showed a stable and well-de?ned redox peak(curve b).The cathodic response resulted from the reduction of Ag+and the anodic process was attributed to the stripping of the silver[18].When CS–MnO2was dropped on a MWNT–Ag-mod?ed electrode,the peak currents decreased(curve c)due to CS–MnO2membrane hindering the transmission of elec-trons.After being modi?ed with nano-Au,the peak currents

Fig.2.TEM images of(A)MWNT–Ag composite and(B)pure MWNT. 176X.Che et al./Journal of Colloid and Interface Science345(2010)174–180

increased and this was attributed to the good conductivity of nano-Au(curve d).Then peak currents decreased step by step when the electrode was modi?ed with anti-AFP and BSA(curve e and curve f),which indicated that anti-AFP and BSA had been immobilized on the electrode surface.After the prepared immunosensor was incu-bated with AFP solution(curve g),a further decrease of the current was observed.This is ascribed to the antigen–antibody complex coating on the surface of the electrode and hindering the transmis-sion of electron toward the electrode surface.

3.4.Optimization of experimental parameters

The pH of the working buffer is an important in?uencing factor for the sensitivity of immunosensors.The effect of pH on the bio-sensor was investigated from4.5to8.0(Fig.6A).The results indi-cated that the current increased from pH 4.5to pH7.0,the maximum response appeared at pH7.0,and then the further increase of pH led to a decrease of peak current.Thus the buffer solution of pH7.0was selected for further experiments.

On the other hand,the in?uence of incubating time is signi?-cant on the reaction of the antibody and antigen.Fig.6B shows the effect of immunochemical incubation time on response signal. The amperometric response of the immunosensor was sharply down with the duration of incubation time in the?rst12min, and then leveled off slowly.Therefore,the incubation time of 12min was adopted in subsequent work.

3.5.Performance of the immunosensor

The BSA/anti-AFP/CS–MnO2/MWNT–Ag/GCE-mod?ed elec-trodes were used to detect AFP.The calibration plots between the changes of cathodic current response(D I)and different con-centrations of AFP are shown in Fig.7A(curves a1and a2).The two linear ranges covered from0.25to12.0and12.0to

Fig.4.SEM images of different membranes:(A)MWNT–Ag;(B)CS–MnO2/MWNT–Ag;and(C)nano-Au/CS–MnO2/MWNT–Ag.

X.Che et al./Journal of Colloid and Interface Science345(2010)174–180177

178X.Che et al./Journal of Colloid and Interface Science345(2010)174–180

250.0ng/ml with correlation coef?cients of0.9984and0.9979, respectively.The detection limit was0.08ng/ml(S/N=3).The CVs of this immunosensor before and after being incubated are shown in Fig.7B.

As a control experiment,the response was also measured on a BSA/anti-AFP/nano-Au/CS/MWNT–Ag-mod?ed electrode without MnO2.As shown in Fig.7A(curves b1and b2),a CS-mod?ed mem-brane electrode exhibited a less steep decline with narrow linear response from0.5to12.0and12.0to250ng/ml.The results indi-cated that CS–MnO2composite?lm enhanced the linear range and sensitivity of the electrode.The reason may be the synergistic ac-tion between MnO2and Ag nanoparticles[26].Moreover,the per-formance of constructed immunosensors was also compared with other immunosensors for the detection of AFP with various meth-ods,as shown in Table1.The data suggested that the current meth-od obviously exhibited a wider linear range and lower detection limit.

3.6.Interference experiments of the immunosensor

Interference experiments were performed to assess whether the immunosensor could respond selectively to different kinds of anti-gens.In this experiment,the immunosensors were incubated with two different solutions:one is AFP standard solution with separate interferent(CEA,IgG,CA125,vitamin C,L-cysteine,and BSA)and the other without.The experimental results indicated that the cur-rent responses in two solutions showed less than5.6%difference. Thus the selectivity of the immunosensor was acceptable.

3.7.Repeatability and stability of the immunosensor

The reproducibility of the proposed immunosensor was deter-mined in50ng/ml AFP solution?ve times.The relative standard deviation(RSD)of the current responses was3.3%.The stability of the immunosensor was also studied in this work.When the immunosensor was cyclically swept for100cycles,a5.3%decrease of the initial response was observed.On the other hand,storage stability of the immunosensor was studied on a30-day period when the electrode was suspended above0.1M PBS(pH7.0)at 4°C.There was no obvious change of current response during the?rst5days.After storage of2weeks and a month,the immuno-sensor retained94.1%and85.3%of its initial current response.The good stability of the immunosensor is attributed to the excellent ?lm forming ability of CS–MnO2/MWNT–Ag composite.Besides,nano-Au deposited on the composite?lm could adsorb molecules ?rmly.

3.8.Real serum sample testing

The proposed immunobiosensor was used to detect real serum samples and the results were compared with those from the typi-cal enzyme-linked immunoassays(ELISA)methods.The results and the relative deviations are shown in Table2.The results indicated that there was no signi?cant difference between the two methods. Thus,the present method may be preliminarily applied for clinical determination of AFP.

4.Conclusion

In this experiment,a new AFP amperometric immunosensor was successfully fabricated by immobilizing anti-AFP on the elec-trode modi?ed with nano-Au/CS–MnO2/MWNT–Ag?lm.The pro-posed immunosensor offers several attractive advantages as follows:?rst,MWNT–Ag composite has great effective surface area and excellent conductivity.Second,CS–MnO2with excellent?lm forming ability could protect MWNT–Ag composite leaking from the electrode.The synergistic action between Ag NPs and MnO2 improves the performance of the immunosensor.Finally,nano-Au provides a congenial microenvironment around anti-AFP mole-cules and greatly ampli?es the coverage.With these advantages, the immobilized technique and the detection methodology would be valuable for detecting other proteins.

Acknowledgments

This work was supported by the National Natural Science Foun-dation of China(20675064),the Ministry of Education of China (Project708073),the Natural Science Foundation of Chongqing City(CSTC-2009BA1003),China and High Technology Project Foun-dation of Southwest University(XSGX02),China.

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Table1

Comparison of different electrochemical immunosensors.

Detection methods Linear

range

(ng/ml)Detection

limit

(ng/ml)

References

Differential pulse voltammetry 1.0–150.00.8[32]

Differential pulse voltammetry0.5–80.00.28[33]

Amperometric?ow/stop-?ow

injection

5.0–150.0 2.0[34]

Cyclic voltammetry0.5–160.00.1[35]

Cyclic voltammetry 1.0–55.00.6[36]

Cyclic voltammetry0.25–250.00.08This work

Table2

Experimental results of different methods obtained in serum samples.

Serum samples12345

Immunosensor(ng/ml) 2.478.3214.727.852.7

ELISA(ng/ml) 2.657.8914.429.351.3

Relative deviation(%)à6.8 5.4 2.1à5.1 2.7

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