ORIGINAL PAPER

Disposable immunoassay for hepatitis B surface antigenbased on a graphene paste electrode functionalizedwith gold nanoparticles and a Nafion-cysteine conjugate

Ke-Jing Huang & Jing Li & Yan-Ming Liu & Xiaoyu Cao &

Sheng Yu & Meng Yu

Received: 13 December 2011 /Accepted: 12 March 2012 /Published online: 23 March 2012# Springer-Verlag 2012

Abstract We report on the modification of a graphene pasteelectrode with gold nanoparticles (AuNPs) and a Nafion-L-cysteine composite film, and how this electrode can serve asa platform for the construction of a novel electrochemicalimmunosensor for the detection of hepatitis B surface anti-gen (HBsAg). To obtain the immunosensor, an antibodyagainst HBsAg was immobilized on the surface of theelectrode, and this process was followed by cyclic voltam-metry and electrochemical impedance spectroscopy. Thepeak currents of a hexacyanoferrate redox system decreasedon formation of the antibody-antigen complex on thesurface of the electrode. Then increased electrochemicalresponse is thought to result from a combination of ben-eficial effects including the biocompatibility and largesurface area of the AuNPs, the high conductivity of thegraphene paste electrode, the synergistic effects of com-posite film, and the increased quantity of HBsAb adsorbedon the electrode surface. The differential pulse voltammet-ric responses of the hexacyanoferrate redox pair are pro-portional to the concentration of HBsAg in the range from0.5–800 ng mL−1, and the detection limit is 0.1 ng mL−1

(at an S/N of 3). The immunosensor is sensitive andstable.

Keywords HepatitisB surface antigen .Gold nanoparticles .

Graphene paste electrode . Nafion/L-cysteine composite .

Immunosensor

Introduction

Nowadays hepatitis B virus (HBV) infection occurs world-wide. It is estimated that more than 300 million people arechronic carriers of the virus [1]. Patients infected with thevirus can develop life-threatening cirrhosis and liver cancer,undergoing great physical pain or mental anguish. Hepatitis Bsurface antigen (HBsAg) can be found in filamentous or spher-ical surface antigen particles in addition to virions. It is gener-ally accepted that the diagnosis of infection by hepatitis B virus(HBV) is based on the presence of the HBsAg in the blood,since that it can generally be detected while still in the incuba-tion period [2]. Hence, developing rapid and sensitive methodsfor measuring HBsAg has great clinical significance in theclinical diagnosis. For proteins quantitative detection, immu-noassay is one of the most important analytical techniques dueto the highly specific binding of antigens and antibodies.Among various immunoassays, electrochemical immunosen-sors have become the predominant analytical technique for thequantitative detection of biomolecules due to their high sensi-tivity, low cost, fast analysis and ease of miniaturization [3].Currently, high sensitivity has become one of the main goals indevelopment of immunoassay methods. Different methods forsignal enhancement have been investigated, such as enzymelabeling [4], rolling circle amplification [5] and nanomateriallabeling [6]. Among these methods, nanomaterial labeling hasgaining growing interest due to the intrinsic advantages ofnanomaterials, such as low cost, good thermal stability andlarge surface area [7].

K.-J. Huang (*) : J. Li :Y.-M. Liu : S. Yu :M. YuCollege of Chemistry and Chemical Engineering,Xinyang Normal University,Xinyang 464000, Chinae-mail: kejinghuang11@163.com

X. CaoSchool of Chemistry and Chemical Engineering,Henan University of Technology,Zhengzhou 450001, China

Microchim Acta (2012) 177:419–426DOI 10.1007/s00604-012-0805-6

Carbon nanomaterials have been widely used in electro-chemical immunosensors, because they usually displaylarge specific surface areas, good conductivity and goodchemical and thermal stability. Graphene is a novel andfascinating carbon nanomaterial, which posses a single layerof carbon atoms in a closely packed honeycomb two-dimensional lattice. Recently, it has attracted increasingattention from researchers because of its unique and excel-lent properties, such as extremely high thermal conductivity,good mechanical strength, high mobility of charge carriers,high specific surface area, quantum hall effect and upstand-ing electric conductivity [8–10]. As electrode materials,graphene can be used for promoting electron transfer be-tween the electroactive species and the electrode and pro-vide a novel method for fabricating chemical sensors orbiosensors [11–13]. On the other hand, gold nanoparticles(AuNPs) have been widely used for the construction ofelectrochemical immunosensors [14, 15] due to the fact thatthey can increase the amount of the biomolecules loadedand then amplify the response.

In this work, we developed a novel electrochemicalimmunosensor for HBsAg based on the advantages of gra-phene, AuNPs and Nafion (Nf)/L-cysteine (L-Cys) compos-ite film. Carbon paste electrode (CPE) is an appealing andwidely used electrode material in the fields of electrochem-ical sensor due to its attractive advantages including simplepreparation, low-cost implementation and renewability [16].The preparation of CPE usually involved in the dispersionof graphite power by a hydrophobic binder to form a ho-mogeneous paste, followed by filling a tube with the result-ing paste [17]. Sometimes, the modifiers including variousbiomolecules, organic compounds and nanoparticles arealso doped into the carbon paste, which makes CPE quitesuitable for bioanalysis. However, the binder used in thepreparation of CPE is usually less or no conducting, whichabsolutely leads to the relatively sluggish electron transferkinetics [18]. Therefore, good-performance modifiers arestill imperative for CPE. Herein, a graphene paste electrode(GPE) was fabricated by the addition of graphene in carbonpaste, and further used as the basal electrode for the elec-trochemical HBsAg immunosensor. The AuNPs preparedby one-step direct chemical reduction were used to immo-bilize the HBsAg antibody (HBsAb). The interactionbetween HBsAb and HBsAg was investigated by the elec-trochemical probe of ferricyanide. Enhanced sensitivity wasachieved by using the large specific surface area of AuNPsto increase HBsAb loading, the high conductivity of GPEand AuNPs to promote electron transfer among probe andthe electrode, which resulted in the high sensitivity of theimmunosensor. Based on signal amplification strategy ofGPE, the fabricated immunosensors using AuNPs aslabels showed a linear response within the wide rangeof 0.5–800 ng mL−1 of HBsAg, low detection limit, good

reproducibility and selectivity, as well as acceptablestability.

Experimental

Materials

Graphite powder, HAuCl4·4H2O and Sodium citrate wasobtained from Jing Wei Chemical Co., Ltd (Shanghai,China, http://www.jingweichem.cn/). Hepatitis B surfaceantibody (HBsAb) and Hepatitis B surface antigen (HBsAg)were purchased fromBosai Chemical Reagent Co. (Zhengzhou,China, http://www.chinabiocell.com/). Carcinoembryonicantigen (CEA), α-Fetoprotein (AFP) prostate-specificantigen (PSA), and human immunoglobulin (HIgG) wereobtained from Sigma (Saint Louis, MO, USA, http://www.sigmaaldrich.com). Phosphate-buffered saline withvarious pH values was prepared with stock standard solutionof Na2HPO4, NaH2PO4 and 0.1 M KCl. All other reagentswere used without any further purification. All the solutionswere prepared with doubly-distilled water.

Instruments

Electrochemical measurements were carried out at aCHI660D electrochemistry workstation (CH InstrumentalCo., ShangHai, China, http://www.instrument.com.cn/netshow/SH101344/). A three-electrode system wasemployed for the electrochemical detection, which wascomposed of a modified GPE as working electrode, a Ptwire as auxiliary electrode and a saturated calomel electrode(SCE) as reference electrode. All the pH values were mea-sured with a PHS-3C precision pH meter (Leici DevicesFactory of Shanghai, China, http://www.lei-ci.com), whichwas calibrated with standard buffer solution every day.

Preparation of graphene

Graphene oxide was firstly synthesized from graphiteaccording to Hummers and Offeman method [19]. Thenthe graphene oxide was reduced to graphene followed atypical procedure: the resulting graphene oxide dispersion(100 mL) was mixed with 70 μL of hydrazine solution(50 wt% in water) and 0.7 mL of ammonia solution(28 wt% in water). The mixture was stirred for 1 h at thetemperature of 95 °C. Finally, black hydrophobic graphenesheets was obtained by filtration and dried in vacuum.

Preparation of gold nanoparticles

AuNPs were prepared by a trisodium citrate reductionmethod as reported before [20]. Briefly, trisodium citrate

420 K. Huang et al.

(5 mL, 38.8 mM) was rapidly added to a boiling solution ofHAuCl4 (50 mL, 1 mM), and the solution was kept contin-ually boiling for another 30 min to give a wine-red solution.After filtering the solution through a 0.45-μm Milliporesyringe to remove the precipitate, the filtrate was stored ina refrigerator at 4 °C.

Fabrication of the immunosensor

The unmodified GPE was prepared by mixing 0.5 mL ofparaffin oil with 256 mg graphite and 64 mg graphenethoroughly in a mortar to form a homogeneous carbon paste.A portion of the carbon paste was filled into one end of aglass tube (i.d.03 mm) and a copper wire was insertedthrough the opposite end to establish an electrical contact.The GPE surface was smoothed on a piece of weighingpaper and then was coated with 5 μL of nafion ethanolsolution (2 %, v/v) and dried in the air. The Nf-modifiedGPE (Nf/GPE) was immersed into L-Cys solution (pH 2.3)for 6 h to obtain L-Cys-Nf/GPE. The obtained L-Cys-Nf/GPE was thoroughly washed with water to remove thephysically absorbed L-Cys. Subsequently, the AuNPs/L-Cys-Nf/GPE was prepared by immersing the L-Cys-Nf/GPE into the prepared Au colloid for 1.5 h and then cleanedwith water and dried under a stream of nitrogen. The AuNPswere adsorbed onto the L-Cys-Nf/GPE by chemisorptionstype interactions between NH2 group and AuNPs. Then themodified electrode (AuNPs/L-Cys-Nf/GPE) was immersedin the HBsAb solution at 4 °C overnight. At last the resultingelectrode was incubated in BSA solution (0.25 %, w/w) about1 h in order to block possible remaining active sites and avoidthe non-specific adsorption. The finished immunosensor(BSA/HBsAb/AuNPs/L-Cys-Nf/GPE) was stored at 4 °Cwhen not in use. Schematic illustration of the stepwise prep-aration process of the immunosensor was shown in Scheme 1.

Experimental measurements

The electrochemical characteristics of the electrode werecharacterized by cyclic voltammetry. After the immunore-action was performed by immersing the immunosensor in

0.01 M phosphate buffer solution (pH 7.0) containing var-ious concentrations of HBsAg for 20 min at 37 °C and thenwashed carefully with double distilled water, the electro-chemical measures were performed in an unstirred electro-chemical cell. The CV measurements were taken in 10 mL0.01 M phosphate buffer solution (pH07.0) 50 mV s−1.Electrochemical impedance spectroscopy measurements werecarried out in the presence of a 5.0 mM [Fe(CN)6]

3-/4-as aredox probe in 0.01 M phosphate buffer solution (containing0.1 M KCl, pH 7.0). The alternative voltage is 5 mV and thefrequency range is 0.1 to 100,000 Hz. The detection is basedon the oxidation peak current response decreasing afterantigen-antibody reaction.

Results and discussion

Characteristics of graphene

The morphology of graphene was characterized by TEM andSEM. The obtained TEM image showed a few layered struc-tures for the graphene, and the transparent sheets were flake-like with wrinkles (Fig. 1a). The SEM image revealed a typicalcrumpled and wrinkled graphene sheet structure (Fig. 1b).

Electrochemical characteristics of electrochemicalimmunosensor

Figure 2 shows the cyclic voltammograms (CVs) of[Fe(CN)6]

3-/4- on different electrodes. A pair of poor redoxpeaks was observed at CPE (Fig. 2a, curve a), which mightbe ascribed to the low conductivity of graphite. While theGPE exhibited well-defined peaks and the peak currentincreased greatly (Fig. 2a, curve b), suggesting the dramaticincrease in the electron transfer rate due to the high conduc-tivity of graphene. The electron transfer of graphene residesfrom its edge rather than its side, where the former actselectrochemically akin to that of edge plane- and the latterto that of basal plane-like sites/defects of highly orderedpyrolytic graphite [21]. The redox peaks decreased dramat-ically after Nf was coated onto the GPE surface owing to the

Nafion L-Cys

ASBbAsBH

GPE

GPE

NH3+

NH3+ NH3

+

GPE

NH3+

NH3+ NH3

+

GPE

AuNPs

GPE

NH3+

NH3+ NH3

+

GPE

NH3+

NH3+ NH3

+Scheme 1 The stepwisefabrication process of theimmunosensor

Disposable immunoassay for hepatitis B surface antigen 421

fact that Nf film greatly obstructs electron and mass transfer(Fig. 2b, curve b). Then the peak currents increased when L-Cys was entrapped in the Nf membrane, forming a layer ofNf-Cys composite membrane that was better for electrontransfer than Nf film solely (Fig. 2b, curve c). A furtherincrease of the peak current (Fig. 2b, curve d) was obtainedafter AuNPs was loaded onto the composite membrane,resulting from the formation of the conductive AuNPsmonolayer. After HBsAb was immobilized on the AuNPs/L-Cys-Nf/GPE surface, the peak currents decreased obvi-ously (Fig. 2c, curve b), which suggested that the proteinHBsAb severely reduced effective area and active sites forelectron transfer. The peak currents decreased in the sameway (Fig. 2c, curve c) after BSA was used to block non-specific sites. Finally, the peak current decreased again afterthe immunosensor was incubated with HBsAg (Fig. 2c,curve d). The reason for this was that the immunocomplexblocked the tunnel for mass and electron transfer.

Electrochemical impedance spectroscopy (EIS) is regardedas an effective method to monitor the interfacial properties ofsurface-modified electrode, making chemical transformationand process on the conductive electrode surface easily under-stood. A typical EIS plot includes a semicircle region and astraight line. The semicircle part, which can be observed athigher frequency, corresponds to the electron-transfer-limited

process, whereas the linear part at the lower frequency rangerepresents the diffusional-limited electron-transfer process.The semicircle diameter equals the electron transfer resistance(Ret). In this study, impedance measurements were performedin 10 mM potassium ferricyanide solution (pH 7.0) containing0.1 M KCl. As shown in Fig. 3, the EIS of GPE displayed alittle semicircle part (curve a), indicating that GPE had a highconductivity. A high interfacial Ret (curve b) was obtained inthe impedance spectrum after the GPE was coated with Nfbecause Nf membrane insulated the conductive support and

Fig. 1 SEM (a) and TEM (b) images of graphene

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Fig. 2 Cyclic voltammograms of different modified electrodes in pH7.0 phosphate buffer solution at 50 mVs-1. a: (a) CPE; (b) GPE. b:GPE (a); Nf/GPE (b); L-Cys-Nf/GPE (c); AuNPs/L-Cys-Nf/GPE (d).c: AuNPs/L-Cys-Nf/GPE (a); HBsAb/AuNPs/L-Cys-Nf/GPE (b);BSA/HBsAb/AuNPs/L-Cys-Nf/GPE (c); HBsAg/BSA/HBsAb/AuNPs/L-Cys-Nf/GPE (d)

422 K. Huang et al.

perturbed the interfacial electron transfer between the elec-trode and the redox indicator in the solution. Then the Retdecreased apparently when L-Cys was embedded into Nf film(curve c), indicating that L-Cys promoted the electron transferand enhanced the conductivity of the electrode. After absorp-tion of AuNPs, a further decrease of Ret was observed (curve d)because of the formation of the conductive AuNPs monolayer.Subsequently, when HBsAb was adsorbed on the surface ofAuNPs, Ret further obviously increased (curve e). The resultwas consistent with the fact that the hydrophobic layer ofprotein further hindered the interfacial electron transfer. TheRet increased in a similar way after BSA was used to blocknon-specific sites (curve f).

Optimizing conditions for immunoassay

The incubation time was an important influence condition ofthe immunosensor [22]. When the analyte antigens reachedthe antibodies on immunosensor surface, it took some timefor the contacting species to form immunocomplex. Figure 4displayed that the current response decreased with the in-crement of incubation time and close to leveled off after20 min in the incubation solution of HBsAg, which impliedthat the building of immunocomplex reached to saturation.Therefore, 20 min was chosen as the incubation time for thedetermination of HBsAg.

Analytical performance of immunosensor

Under optimized conditions, differential pulse voltammetry(DPV) was employed to investigate the immunoreactionbetween the immobilized HBsAb and HBsAg. As can beseen from Fig. 5, the DPV peak currents showed an increase

with the increment of HBsAg concentration. As presented inthe Fig. 5, the decrease of DPV peak current was propor-tional to the logarithm of HBsAg concentration in the rangeof 0.5 to 800 ng mL−1 with a regression equation of the formΔIpa (μA)046.245+38.697 logC (ng mL−1) (R00.991).The detection limit was 0.1 ng mL−1 (S/N03).

A comparison of the detection methods were shown inTable 1, which included the limit of detection and the linearrange. Table 1 indicated that the developed immunosensor(BSA/HBsAb/AuNPs/L-Cys-Nf/GPE) exhibited lower detec-tion limit and wider measurement range. The reason might beas follows: firstly, the excellent electrical conductivity of GPE

0 1500 3000 4500 6000 7500 9000 10500

200 400 600 800

Zim

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)

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hm)

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Fig. 3 EIS of GPE (a); Nf/GPE (b); L-Cys-Nf/GPE (c); AuNPs/L-Cys-Nf/GPE (d); HBsAb/AuNPs/L-Cys-Nf/GPE (e); BSA/HBsAb/AuNPs/L-Cys-Nf/GPE (f) in 5 mM Fe(CN)6

3-/4- solution containing0.1 M KCl

5 10 15 20 25 30

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I / μ

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Fig. 4 Influence of incubation time (a), incubation temperature (b)and pH (c) on the response signals

Disposable immunoassay for hepatitis B surface antigen 423

enhanced the charge transport; secondly, the formation of theL-Cys-Nf film increased the immobilization amount ofAuNPs. The unique physical and chemical features couldincrease the surface loading amount of the HBsAb.

Selectivity of the immunosensor

Nonspecific adsorption is the major problem in label-freeimmunosensing. To investigate the specificity of the devel-oped immunosensor, 100 ng mL−1 of CEA, PSA, AFP andHIgG, were used in this study. Current responses of thedeveloped immunosensor in 1.0, 10, 100, 400, and800 ng mL−1 HBsAg solutions containing interfering sub-stances with different concentrations were assayed, and theCV values were 2.6–3.5 %, 2.0–3.8 %, 1.8–3.9 %, 1.7–3.2and 2.3–3.5 %, respectively. So the selectivity of the devel-oped immunosensor was acceptable.

Reproducibility and stability of the immunosensor

The reproducibility of the resulted immunosensor was in-vestigated at HBsAg concentration of 20 ng mL−1. Six

immunosensors that made independently showed anacceptable relative standard deviation (RSD) of 4.8 %, sug-gesting that the electrode-to-electrode reproducibility of thefabrication protocol was satisfactory. The stability of theimmunosensor was also studied in this work. When theimmunosensor was cyclically swept for 100 cycles, a4.2 % decrease of the initial response was observed. Onthe other hand, storage stability of the immunosensor wasstudied on a 6-month period. The relative change in currentresponse was tested at the same HBsAg concentration of20 ng mL−1 every 10 days. The current response maintainedabout 90.2, 85.8 %, 83.3 %, 82.1 %, 81.6 % and 80.1 % ofthe original value after the storage periods of 1 month,2 months, 3 months, 4 months, 5 months and 6 months,respectively, indicating the effective retention of the activityof the immobilized HBsAb. The good stability of proposedimmunosensor maybe contributed to following two factors.On the one hand, the gold nanoparticles provided a biocom-patible microenvironment around the biological molecules.On the other hand, the protein molecules were attachedfirmly onto the surface of composite matrix.

Preliminary application of the immunosensor

In order to demonstrate the use of the fabricated immuno-sensor for the determination of the HBsAg in human serum,six serum samples from the hospital affiliated to our univer-sity were examined by the developed electrochemicalimmunosensor and the enzyme-linked immunosorbentassays (ELISA) methods, respectively. Table 2 showed the

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Fig. 5 Calibration plot of the immunosensor for detection of HBsAg.Insets: DPV responses toward various concentrations of HBsAg

Table 1 Comparison ofdifferent electrochemicalimmunosensors for thedetermination of HBsAg

Modified electrode Linear range(ng mL−1)

Detection limit(ng mL−1)

Reference

HBsAb-polyvinyl butyral-AuNPs 8.0–1280 2.3 [23]

HBsAb-polyvinyl butyral-AuNPs/Pt 10–160 7.8 [24]

HBsAb-chitosan/SiO2/ITO 6.85–708 3.89 [25]

HBsAb-AuNPs/thionine/Nafion 2.56–563.2 0.85 [26]

HBsAb-AuNPs/Prussian blue 2.13–314.3 0.42 [27]

HRP-HBsAb/AuNPs/Al2O3/RTIL/Nafion/Au 1.2–430 0.3 [28]

Poly(allylamine)-ferrocene/AuNPs/HBsAb/GCE 0.1–150 0.04 [29]

HBsAb/AuNPs/L-Cys-Nf/GPE 0.5–200 0.01 This work

Table 2 Experimental results of two methods obtained in serumsamples

Samples 1 2 3 4 5

ELISA(ng mL−1)

20.1±0.2 22.3±0.1 35.6±0.3 54.2±0.7 73.5±0.6

This method(ng mL−1)

18.6±0.2 23.1±0.6 37.1±0.4 57.9±0.2 71.4±0.3

Relativedeviation (%)

−7.5 3.6 4.2 6.8 −2.9

424 K. Huang et al.

results of the two methods studied. It can be seen that therelative error between the two methods was from −7.5 % to6.8 %, suggesting an acceptable agreement. Hence, thedeveloped immunoassay might provide a feasible alterna-tive tool for determining the HBsAg in human serum in aclinical laboratory.

Conclusions

In this paper, a novel strategy of an amperometric immuno-sensor for HBsAg was developed based on the HBsAbadsorbed in AuNPs/L-Cys-Nf/GPE. The advantages of theimmunosensor are as following: (1) the appearance of gra-phene in the CPE with high conductivity greatly promotedthe direct electron transfer between [Fe(CN)6]

3-/4- and theunderlying electrode; (2) the developed strategy offered asimple and convenient methodology for the preparation ofstable structured L-Cys-Nf composite to increase the ad-sorption amount of AuNPs; (3) AuNPs adsorbed in L-Cys-Nf composite increased the conductibility, biocompatibilityand the immobilization amount of HBsAb. On the basis ofthe above reasons, the prepared immunosensor exhibitedhigh stability and a low detection limit. The simplicity infabrication procedures, ease of the detection step and goodreproducibility of the developed method opened up an in-creasing possibility for the future development of practicaldevices for determination of HBsAg and other proteins.

Acknowledgments This work was supported by the NationalNatural Science Foundation of China (20805040), Program for Science& Technology Innovation Talents in Universities of Henan Province(2010HASTIT025), Excellent Youth Foundation of He’nan ScientificCommittee (104100510020).

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