Journal of Electroanalytical Chemistry 655 (2011) 50–55

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Journal of Electroanalytical Chemistry

journal homepage: www.elsevier .com/locate / je lechem

Label-free electrochemical detection of cancer marker based on graphene–cobalthexacyanoferrate nanocomposite

Ting Li a, Minghui Yang b,⇑, He Li c,⇑a Department of Surgery, The Second Xiang-ya Hospital, Central South University, Changsha 410011, PR Chinab College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, PR Chinac School of Medical and Life Sciences, University of Jinan, Jinan 250022, PR China

a r t i c l e i n f o

Article history:Received 26 October 2010Received in revised form 28 January 2011Accepted 12 February 2011Available online 17 February 2011

Keywords:Label-freeProstate specific antigenGrapheneSynergistic effectsImmunosensor

1572-6657/$ - see front matter � 2011 Elsevier B.V. Adoi:10.1016/j.jelechem.2011.02.009

⇑ Corresponding authors. Tel./fax: +86 731 888363E-mail addresses: ymhhaohao@gmail.com (M. Yang

a b s t r a c t

The synergistic effect between graphene sheet (GS) and cobalt hexacyanoferrate nanoparticle (CoNP) wasinvestigated, and showed that the electroactivity of CoNP was greatly enhanced in the presence of GS dueto great electron-transfer ability of GS. A label-free electrochemical immunosensor for the sensitivedetection of prostate specific antigen (PSA) was fabricated. Molecule 1-pyrenebutanoic acid, succinimidylester (PBSE) was adsorbed onto GS and the colloidal solution containing GS–CoNP–PBSE was added ontoglassy carbon electrode surface to form stable thin film with high electroactivity. After anti-PSA antibodywas conjugated onto PBSE, the modified electrode could be used as an amperometric immunosensor forthe detection of PSA. The specific antibody–antigen immunocomplex formed on the electrode resulted inthe decrease of amperometric signal of the electrode. The amperometric signal decreased linearly withPSA concentration in the range of 0.02–2 ng/mL with a low detection limit of 0.01 ng/mL. The immuno-sensor has the advantages of high sensitivity, good selectivity and stability, and could become a promis-ing technique for cancer marker detection.

� 2011 Elsevier B.V. All rights reserved.

1. Introduction

The early detection of tumor markers plays an important role inclinical research and early diagnosis of cancer [1–4]. Due to thespecific binding of antibody to its corresponding antigen, immuno-sensors based on antibody–antigen interaction are one of the mostwidely used analytical techniques in the quantitative detection oftumor markers [5–7]. Typically, a sandwich-type protocol wasused to prepare the immunosensor with the primary antibodyimmobilized onto the solid surface, specific antigen bound to theantibody site and labeled secondary antibody bound to the antigen[8–10]. In recent years, different probes have been investigated aslabels for such immunosensors including enzymes, metal nanopar-ticles, carbon nanotube, and fluorescence dyes in order to amplifythe signal [11–13]. However, the labeling process is time-consum-ing, costly and easy to be denatured due to the susceptibility ofbiomolecules.

Besides of the sandwich protocol, the label-free immunosensorsbased on direct detection of antibody–antigen binding have gainedgrowing interest recently. Methods including electrochemistry,surface plasmon resonance (SPR), piezoelectric and cantilever havebeen employed to prepare label-free immunosensors [14–17].

ll rights reserved.

76.), chm_lih@ujn.edu.cn (H. Li).

Among these methods, electrochemical immunosensor due to itshigh sensitivity, low cost, and ease of miniaturization has becomethe predominant analytical technique for quantitative detection ofbiomolecules. Zhu et al. reported a label-free electrochemilumines-cence (ECL) immunosensor by combining CdSe nanocrystals,carbon nanotube and chitosan film. The composite-film-modifiedelectrode showed high ECL intensity; and after antibody wasbound to the film, specific immunoreaction between antibodyand antigen resulted in the decrease of ECL intensity, which wasused for the detection of antigen [18]. Xia et al. reported a label-free amperometric immunosensor based on chitosan–ferrocenecomposite film for the detection of hepatitis B surface antigen, inwhich the hepatitis B surface antigen concentration was measuredthrough the decrease of amperometric response in the correspond-ing specific binding of antigen to antibody [19].

Since the discovery of carbon nanotube in 1991, carbon basednanomaterials including carbon nanotube, carbon nanofibers andhighly ordered mesoporous carbon have been widely used in prep-aration of modified electrodes [20–22]. Recently, graphene sheet(GS), a single layer of carbon atoms bonded together in a hexagonallattice, has stimulated a vast amount of research due to its fascinat-ing properties [23–25]. It has large specific surface area, good con-ductivity and fracture strength. Additionally, GS is considered asthe basic building block that can be wrapped into 0D fullerenes,rolled into 1D carbon nanotube and stacked into 3D graphite[26]. Therefore, GS is promising candidates as components in

T. Li et al. / Journal of Electroanalytical Chemistry 655 (2011) 50–55 51

applications such as energy-storage materials, polymer compos-ites, liquid crystal devices and mechanical resonators.

The synergistic action between carbon nanotube and redoxmediators to facilitate electron-transfer process has been widelystudied. Zhang et al. reported the integration between carbonnanotube and toluidine blue, which resulted in remarkableimprovement of the electrocatalytic activity of toluidine bluetowards NADH [27]. Li et al. reported the construction of ampero-metric biosensors via grafting Prussian blue (PB) nanoparticles onthe polymeric matrix of multiwalled carbon nanotubes (MWCNTs)and poly(4-vinylpyridine) (PVP) [28]. The electrode modified withthe MWCNT/PVP/PB composite film shows prominent electrocata-lytic activity toward the reduction of hydrogen peroxide, whichcan be explained by the remarkable synergistic effect of theMWCNTs and PB. However, there are still few reports about suchsynergistic action between GS and other materials [29]. In thispaper, cobalt hexacyanoferrate nanoparticle (CoNP) was selectedand combined with GS, the electroactivity of CoNP was greatly en-hanced. Based on this finding, a label-free immunosensor for thesensitive detection of prostate specific antigen (PSA) was prepared.After antibody was linked to the GS–CoNP thin film through mole-cule 1-pyrenebutanoic acid, succinimidyl ester (PBSE), the anti-body–antigen immunocomplex formed on the electrode surfaceresulted in the decrease of amperometric signal. The immunosen-sor provided a simple, economic, sensitive and specific method forcancer marker detection, which could find potential application inclinical analysis.

2. Materials and methods

2.1. Apparatus and reagents

Graphite was purchased from Shanghai Carbon Co., Ltd. (Shang-hai, China). 1-pyrenebutanoic acid, succinimidyl ester (PBSE) wasobtained from Fisher Scientific. Prostate specific antigen (PSA)and anti-PSA antibody were purchased from Dingguo BiochemicalReagents (Beijing, China). All other chemicals were of analytical re-agents grade and used without further purification. Phosphate buf-fered saline (PBS, 0.1 M containing 0.1 M NaCl, pH 7.4) was used aselectrolyte for all electrochemistry measurement. Double distilledwater was used throughout the experiments.

All electrochemical measurements were performed on a CHI760D electrochemical workstation (Shanghai CH Instruments Co.,China). Transmission electron microscope (TEM) images wereobtained from a JEOL JEM-2010 microscope (Japan). A conventionalthree-electrode system was used for all electrochemical measure-ments: a glassy carbon electrode (GC, 3 mm in diameter) as theworking electrode, an Ag/AgCl electrode as the reference electrode,and a platinum wire electrode as the counter electrode.

2.2. Preparation of graphene sheet (GS)

GS was prepared from graphite oxide (GO) through a thermalexfoliation method [30]. At first, GO powders were produced fromgraphite by a modification of Hummer’s method [31]. In a typicalexperiment, 5 g of graphite was oxidized by reacting with100 mL of concentrated H2SO4 under stirring for 12 h. Then, whileimmersing the reaction vessel in an ice bath, 30 g of KMnO4 wasadded slowly. After the addition of KMnO4, the solution was stirredat 100 �C for another 12 h to fully oxidize graphite GO. The ob-tained GO was then thoroughly washed and dried.

Thermal exfoliation of GO was achieved by placing GO (100 mg)into a quartz tube under argon atmosphere. The quartz tube wasflushed with argon for 10 min, and then quickly inserted into a fur-nace preheated to 1000 �C and held in the furnace for about 1 min.

2.3. Preparation of cobalt hexacyanoferrate nanoparticles (CoNP)

CoNP nanoparticle was synthesized according to protocolreported previously [32]. Typically, 35 mL of a 0.01 M aqueoussolution of CoCl2 was added dropwisely to a 35 mL solution of0.05 M K3Fe(CN)6 containing 0.05 M KCl under stirring. After com-plete addition, the liquid was vigorously agitated for 5 min andthen immediately subjected to filtration. The precipitate was con-tinuously washed with water and then collected after filtration anddried overnight at room temperature to give a quantitative powdercompound.

2.4. Modification of the electrodes

GC electrode was polished with 1, 0.3, and 0.05 lm aluminapowder sequentially and then washed ultrasonically in ethanoland water for a few minutes, respectively. To prepare theGS–CoNP–PBSE composite-film-modified electrode, 1 mg of PBSEwas dissolved in 1 mL water, and different concentrations of GSand CoNP were added into the solution. After sonication for5 min, 5 lL of the mixture was dropped onto the electrode surfaceand dried. The electrode was then thoroughly rinsed with PBS toremove the unbounded particles.

To immobilize the antibody onto electrode surface, 5 lL of anti-PSA antibody solution (10 lgmL) was added onto electrode surfaceand incubated for 1 h. The electrode was then washed and incu-bated in 1% bovine serum albumin (BSA) solution for another30 min to block nonspecific binding sites. Afterwards, the electrodewas incubated with different concentrations of PSA solution andafter washing, the immunosensor is ready to measure. Fig. 1 out-lines the fabrication scheme of the immunosensor, which includesthe formation of GS–CoNP composite film on GC electrode, thelinkage of molecular PBSE, the immobilization of antibody andthe specific immunoreaction.

3. Results and discussion

3.1. Characterization of GS–CoNP film

The GS was first characterized by TEM. As shown in Fig. 2A,large GS, with wrinkled paper-like structure was observed. Theyare transparent with irregular size. Fig. 2B shows the TEM imageof the as-synthesized CoNP, which shows that the particles aresphere shape. The diameter of the particles is around 30 nm, withsome larger ones reaching around 50 nm.

To discern the role of GS and CoNP in the composite film andpossible synergy between them, three kinds of nanocompositematerials were used to prepare the electrode: GS–PBSE, CoN-P–PBSE and GS–CoNP–PBSE, and the performances of the elec-trodes were compared. Molecule PBSE was selected as it can beeasily adsorbed onto GS through k-k stacking and the succinimidylester can then be used for the later conjugation of biomolecules. Toprepare the electrodes, 5 lL of each nanocomposite solution wasdropped onto the surface of the electrode. After dried, the cyclicvoltammograms (CVs) of the prepared electrodes in PBS solutionwith scan rate of 0.1 V/s were recorded. As shown in Fig. 3A, theCoNP–PBSE (1 mg/mL of CoNP) modified electrode exhibit two setsof well-defined reversible redox peaks (curve a), which couldascribe to the transformations between Fe(II) and Fe(III) inCo3

II[FeIII(CN)6]2 and Co2IIFeII(CN)6 [33,34]. Their formal potential,

evaluated as the mean of the anodic and cathodic potentials, are+0.402 and +0.835 V, respectively. While keep the concentrationof CoNP unchanged, with the introduction of GS, there is a sharpincrease of the peak current for GS–CoNP–PBSE modified electrode(curve b, 0.5 mg/mL GS and 1 mg/mL CoNP). Further increase of the

GC GC

1-Pyrenebutanoic acid, succinimidyl ester

Antibody Antigen

GC

Amperometric signal

O

O N

O

O

OO N

O

OOO N

O

O

H2NO

HN

OHN

= GS

= CoNP

Fig. 1. Fabrication steps of the immunosensor.

Fig. 2. TEM images of graphene sheet (A) and cobalt hexacyanoferrate nanoparticle (B).

52 T. Li et al. / Journal of Electroanalytical Chemistry 655 (2011) 50–55

GS concentration, the peak current increased again (curve c, 1mg/mL GS and 1 mg/mL CoNP). For the GS–PBSE modified elec-trode, no obvious voltammetric peaks were observed (the insert).The fact that there was virtually no change in the shape or peak po-tential of the CVs suggests that the introduction of GS did not affectthe electron-transfer process between the CoNP and electrode. Inparticular, the introduction of 1 mg/mL of GS amplified the peakcurrent of CoNP by �6 times, which revealed the presence of syn-ergistic effects in GS–CoNP film leading to improved electroactivityof CoNP [28]. The synergistic effects due to GS could be ascribed tothe good electron-transfer ability of GS.

The stability of the GS–CoNP–PBSE film modified electrode wasalso tested as shown in Fig. 3B. The electrode was scanned success-fully for 20 cycles in PBS at 0.1 V/s, no observable change of peakcurrent and position was found, indicating good stability of theelectrode.

3.2. Characterization of the immunosensor

Since we have proved the GS–CoNP–PBSE film modified elec-trode has good stability and high electroactivity, a label-freeimmunosensor for the detection of PSA was prepared. Anti-PSA

antibody was directly conjugated onto PBSE based on the succin-imide ester [35] and the large specific surface area of the GS wasused to increase the antibody loading. In order to characterize thefabrication process of the immunosensor, CVs at each immobiliza-tion step was recorded and shown in Fig. 4. When antibody wasconjugated onto the electrode, there is an obvious decrease of theamperometric current (curve b). The reason for the decrease ofthe current maybe that the oxidation/reduction electrochemicalprocess of CoNP is accompanied by a flux of cations from theelectrolyte solution to maintain electron-neutrality [36,37], sowhen the antibody was on the electrode surface, it acted as theelectron communication and mass-transfer blocking layer, whichprevented the access of cations to the CoNP and then the oxida-tion/reduction current of CoNP was decreased. Finally, when1 ng/mL of PSA was captured, the amperometric currentdecreased again, indicating the formed immunocomplex afterspecific immunoreaction greatly inhibited the reaction on theelectrode surface. From these results, it can be seen that theGS–CoNP–PBSE nanocomposite modified electrode can be usedfor the detection of PSA. The largest peak current change, whichis the current change of the oxidation peak at +0.416 V was usedas signal to different concentration of PSA.

-0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2-30

-20

-10

0

10

20

30A

-0.4 0.0 0.4 0.8 1.2-1.6-0.80.00.81.6

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-10

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40B

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rent

, μΑ

Potential,V

Fig. 3. (A) Cyclic voltammograms (CVs) of the GS–CoNP–PBSE film modified glassycarbon electrode in PBS with 1 mg/mL of CoNP and different concentration of GS:(a) 0, (b) 0.5 and (c) 1 mg/mL. (B) 20 successive scans of the GS–CoNP–PBSE filmmodified electrode in PBS. Scan rate: 0.1 V/s.

-0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2-30

-20

-10

0

10

20

30

cb

a

Cur

rent

, μΑ

Potential, V

Fig. 4. CVs of (a) GS–CoNP–PBSE, (b) GS–CoNP–PBSE/Ab and (c) GS–CoNP–PBSE/Ab/PSA modified electrode in PBS. Scan rate: 0.1 V/s.

0.0 0.5 1.0 1.5 2.0

1

2

3

4

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7

8A

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rent

, μΑ

Concentration of GS, mg/mL

0.0 0.5 1.0 1.5 2.01

2

3

4

5

6

7

8B

Cur

rent

, μΑ

Concentration of CoNP, mg/mL

Fig. 5. Effect of the concentration of GS (A) and CoNP (B) on the response of theimmunosensor to 1 ng/mL PSA. Error bar = RSD (n = 5).

-0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2-20

-10

0

10

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0.0 0.5 1.0 1.5 2.002468

101214

cb

a

Cur

rent

cha

nge,

μΑ

Concentration of PSA, ng/mL

e

a

Cur

rent

, μΑ

Potential, V

Fig. 6. CVs of the GS–CoNP–PBSE modified immunosensor in the presence ofdifferent concentrations of PSA. (a) 0, (b) 0.1, (c) 1, (d) 10, and (e) 50 ng/mL. Scanrate, 0.1 V/s. The insert is calibration curves of the (a) GS–CoNP–PBSE, (b) CoNP–PBSE and (c) GS–PBSE modified immunosensor to different concentration of PSA.Error bar = RSD (n = 5).

T. Li et al. / Journal of Electroanalytical Chemistry 655 (2011) 50–55 53

The effect of the concentration of GS and CoNP in the GS–CoNP–PBSE film on the response of the immunosensor towards PSA wasinvestigated. As shown in Fig. 5A, with the increasing of the GSconcentration from 0 to 1 mg/mL, the current response of the elec-trode to 1 ng/mL PSA was increased gradually, and reached themaximum at 1 mg/mL. We think the increase of sensitivity is be-cause the increase of GS concentration improved the electroactiv-ity of CoNP, which helped to improve the sensitivity. Furtherincrease the GS concentration from 1 to 2 mg/mL resulted in the

decrease of the sensitivity of the immunosensor. This might bethe reason that with further increase of GS in the modifying layer,excessive GS may be stacked each other. This process will entrapCoNPs between the GS layer, decreasing the peak current. So1 mg/mL of GS concentration was selected as optimal conditionto prepare the immunosensor. Similar results were obtained forthe concentration of CoNP and also a 1 mg/mL of CoNP concentra-tion was selected (Fig. 5B).

Fig. 6 shows the CVs of the immunosensor before and afterreacting with different concentrations of PSA. The amperometric

2 4 6 8 100

2

4

6

8

10

Imm

unos

enso

r re

sults

, ng/

mL

ELISA results, ng/mL

Fig. 8. Compare of the PSA concentrations in serum samples determined with theproposed immunosensor and the ELISA method. Error bar = RSD (n = 5).

54 T. Li et al. / Journal of Electroanalytical Chemistry 655 (2011) 50–55

signal of the oxidation peak at +0.416 V in the presence of PSA(curve b–e) was lower than that in the absence of PSA (curve a)and decreased gradually with the increase of the concentration ofPSA, which suggest that different PSA concentration could bedetermined.

The insert of Fig. 6 shows under the optimum conditions, thecurrent change of the peak at +0.416 V to different concentrationof PSA (curve a). The calibration curve showed good linear relation-ships between the current change and the concentration of PSA inthe range from 0.02 ng/mL to 2 ng/mL (curve a). Based on S/N = 3, adetection limit of 0.01 ng/mL was obtained. The low detection limitmay be attributed to two factors. First, the large specific area of GScan be used to adsorb a large amount of PBSE molecule, and thenthe amount of antibodies conjugated onto GS was greatly im-proved. Secondly, the GS–CoNP–PBSE nanocomposite immobilizedon the GC electrode produced a thin film with relatively high elec-troactivity, which increased the sensitivity.

To further confirm the advantages of the GS–CoNP–PBSE film to-ward the detection of PSA, control experiment using CoNP-BPA andGS–BPA was also studied. For the CoNP-BPA modified electrode, thecurrent change due to the same concentration of PSA was about just10% of that of GS–CoNP–PBSE (Fig. 6, insert, curve b), with a rela-tively narrow linear range from 0.2 ng/mL to 2 ng/mL. While forthe GS–BPA modified electrode, almost no current change was foundtoward PSA (Fig. 6, insert, curve c).

3.3. Selectivity, reproducibility, stability, and regeneration of theimmunosensor

To evaluate the reproducibility of the immunosensors, a seriesof five electrodes were prepared for the detection of 1 ng/mLPSA. The relative standard deviation (RSD) of the measurementsfor the five electrodes was 6.7%, suggesting the precision andreproducibility of the proposed immunosensor was quite good.

The selectivity of the immunosensors was also tested. Theamperometric response of the immunosensor toward human IgG,BSA and glucose were studied. The 1 ng/mL of PSA solution con-taining 100 ng/mL of interfering substances (human IgG, vitaminC, BSA, glucose) was measured by the immunosensor and theresults are shown in Fig. 7. The current variation due to the inter-fering substances was less than 8% of that without interferences,indicating the selectivity of the immunosensor was acceptable.

To test the stability of the immunosesnor, the sensor was storedin pH 7.4 PBS when not in use. After three weeks, no apparentchange for the detection of the same PSA concentration was found.

1 2 3 4 5

2

4

6

8

10

Cur

rent

, μΑ

Samples

Fig. 7. Amperometric response of the immunosensor to 1 ng/mL PSA (1), 1 ng/mLPSA + 100 ng/mL BSA (2), 1 ng/mL PSA + 100 ng/mL human IgG (3), 1 ng/mLPSA + 100 ng/mL glucose (4) and 1 ng/mL PSA + 100 ng/mL vitamin C(5). Errorbar = RSD (n = 5).

The good stability can be ascribed to two reasons: the first is thatthe stability of the GS–CoNP–PBSE film is good, the second is thatthe antibody could firmly attach to the electrode surface throughcovalent binding.

To test the possibility of regenerating the immunosensor, 0.2 Mglycine–hydrochloric acid (pH 2.0) was used to wash the electrode.After the detection of 1 ng/mL PSA, the immunosensor was im-mersed into the glycine-hydrochloric acid for 1 min to break theantibody–antigen linkage, and then used to detect 1 ng/mL PSAagain. After five regeneration cycles, the immunosensor retainedabout 90% of it original value, and a RSD of 7.9% was obtained.

3.4. Real sample analysis

The immunosensor was used for real sample analysis, six clini-cal serum samples were tested and the results were comparedwith those obtained by standard ELISA methods. There is no signif-icant difference between the results of the two methods with rel-ative errors less than 9%, indicating the proposed method couldbe used for routine clinical test (Fig. 8).

4. Conclusions

This paper reported a novel and simple label-free electrochem-ical immunosensor for the detection of prostate cancer marker PSA.CoNP was prepared and in combination with GS, the electroactivityof CoNP was greatly improved due to the synergistic effect.Molecule PBSE was selected and adsorbed onto GS through p–pstacking, which was then used for the conjugation of anti-PSAantibody. The GS–CoNP–PBSE composite film displayed highelectroactivity and good stability. After the antibody–antigenimmunocomplex was formed on the electrode surface, there is adecrease of amperometric signal which is linear with PSA concen-tration in the range of 0.02–2 ng/mL. This immunosensor exhibitedsatisfactory selectivity, reproducibility and stability. We believethat this simple electrochemical immunosening platform has po-tential application for further development in practical cancerdiagnosis system.

Acknowledgements

This study was supported by the Natural Science Foundation ofChina (No. 21075052, 81000976), the Natural Science Foundationof Shandong Province (No. ZR2010BM030, ZR2010BQ010), the Sci-ence and Technology Key Plan Project of Shandong Province (No.2010GSF10628), and the Science and Technology DevelopmentPlan Project of Jinan City (No. 201004015).

T. Li et al. / Journal of Electroanalytical Chemistry 655 (2011) 50–55 55

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