ORIGINAL PAPER

Electrocatalytic oxidation of bilirubinat ferrocenecarboxamide modified MWCNT–goldnanocomposite electrodes

Cong Wang & Guangfeng Wang & Bin Fang

Received: 25 November 2007 /Accepted: 17 March 2008 /Published online: 15 April 2008# Springer-Verlag 2008

Abstract A glassy carbon electrode (GCE) modified withferrocenecarboxamide, gold nanoparticles and multiwallcarbon nanotubes was constructed and characterized byfield emission scanning electron microscopy and cyclicvoltammetry. The electrochemical behavior of bilirubin(BR) on the modified electrode was investigated and it wasfound that the modified electrode had an obvious electro-catalytic effect on bilirubin. Compared with a bare GCE,the modified electrode exhibited a marked enhancement inthe current response for bilirubin. Amperometry wasemployed to investigate the electrocatalytical oxidation ofbilirubin on the modified electrode. As a result, it exhibitsan excellent electrocatalytic response to bilirubin with aresponse time of less than 5 s, a broad linear range of 1 to100 μmol L−1, as well as good long-term stability andreproducibility.

Keywords Ferrocenecarboxamide . Gold nanoparticle .

Multiwall carbon nanotubes .Modified electrode .

Bilirubin . Electrocatalytical oxidation

Introduction

Bilirubin is one of the various bile pigments resulting fromthe metabolic breakdown of blood haeme [1]. Serumbilirubin levels are clinically determined as part of theroutine in newborn nurseries. This is because highconcentration of bilirubin in the blood may cause braindamage or even death, and this is especially the case inbabies [2]. So it is practically significative to research themethod for the determination of bilirubin. Current bilirubinanalyses are based either on direct spectroscopic measure-ments [1, 3] or on colourimetric measurements followingdiazotization of the analyte. Compared with the abovemethods, the electrochemical method is more simple andconvenient. Bilirubin, itself, exhibits electrochemical activ-ity, but several problems are encountered during thedevelopment of biosensor for bilirubin [2, 4–7].

Bilirubin oxidase is a multicopper oxidase whichcatalyses oxidation of bilirubin to biliverdin with com-bined reduction of dioxygen to water. It was also shownthat BOD is able to electrocatalyze oxygen reduction inproximity of neutral pH. This is crucial for the develop-ment of oxygen-reducing cathodes for biofuel cellsoperating under physiological conditions [8]. However,some chemical redox mediators have to be used in order toestablish electrochemical communication between theenzyme and an electrode, which makes it difficult for invivo application [9].

In recent years, ferrocene (Fc) derivatives have attractedcontinuous and increasing interest in many areas because oftheir unique optical, thermal and redox behavior. One of themost important properties is their redox behavior. So theyhave been widely used as electron transfer mediators tomodify electrodes. However, they cannot be adsorbedstrongly on the electrode surface, so the electrode direct

Microchim Acta (2009) 164:113–118DOI 10.1007/s00604-008-0041-2

C. Wang :G. Wang :B. Fang (*)School of Chemistry and Materials Science,Anhui Normal University,Wuhu, Anhui 241000, People’s Republic of Chinae-mail: wangyusandra@yahoo.com.cn

C. WangAnhui Key Laboratory of Spin Electron and Nanomaterials(Cultivating Base), Department of Chemistry and Biology,Suzhou College,Suzhou, Anhui 234000, People’s Republic of China

C. Wang :G. Wang :B. FangAnhui Key Laboratory of Functional Molecular Solids,Wuhu, Anhui 241000, People’s Republic of China

modifying is unstable. Various methods have developed toimprove the attachment between Fc derivatives and theelectrode surface [10–15].

Metal nanoparticles are very increasing for the electrodemodification owing to their extraordinary catalytic activitiesover corresponding bulk metal electrodes [16–23]. Goldnanoparticle (NG) is one of the most intensively studiedand applied metal nanoparticles in electrochemistry owingto its stable physical and chemical properties, catalyticactivity and small dimensional size. These unique proper-ties allow them to provide some important functions ofelectroanalysis and the construction of electrochemicalsensors [24, 25].

The importance of the interactions between carbonnanotubes (CNTs) and redox-active metal nanoparticleshas been increasingly recognized, which is an area ofintense research because of their remarkable properties [26,27]. Recent studies proved that the complexes of CNTs andmetal nanoparticles had been formed [26–29]. And as aresult of unique tubular structure, carbon nanotube ismolecular-scale wire with high mechanical stiffness andstrength. So here we wish to report the development of abilirubin biosensor based on modified electrode thatconsists of a multilayer network of NG, multiwall carbonnanotubes (MWCNTs) and ferrocene derivative.

The configuration of immobilized ferrocene derivativeon NG/MWCNTs matrix is obviously important to theefficiency of catalytic activities, which is obviously animportant factor for configuration of sensors. In this paper,with the NG/MWCNTs hybrid as the immobilizationmatrix, and the Ferrocenecarboxamide(FcAI, the chemicalstructure is as Scheme 1) as an ideal electron transfermediator, a novel biosensor based on multilayer filmscomposed of MWCNTs, NG and FcAI on the glassy carbonelectrode is developed. The result shows that modifiers

have strong attachment to the electrodes surface and highchemical stability in air. And then the electrochemicalbehavior of the modified electrodes is investigated.

Experimental

Chemicals and reagents

HAuCl4·3H2O was purchased from Acros (http://www.reagent.com.cn). Bilirubin was purchased from Fluka(http://www.reagent.com.cn). The acid treated Multiwallcarbon nanotubes (95% purity) were purchased fromChengdu Institute of Organic Chemistry of Academy ofSciences and synthesized by chemical vapor deposition.Ferrocenecarboxamide (C11H11FeNO, CAS number: 1287-17-8) was purchased from Weite Chemical Plant. All otherchemicals were of analytical grade and were used withoutfurther purification. All the solutions were prepared withdoubly distilled water with a quartz apparatus. Due to thelow solubility of bilirubin in acidic solution in water, allmeasurements were performed in 0.05 mol L−1 Tris buffer(pH=8.0), and all bilirubin solutions were wrapped inaluminium foil to avoid light exposure [2]. GCE (typeCHI104, effective diameter 3 mm) was purchased fromChenHua instruments Co. CHI, Shanghai, China.

Apparatus and measurements

All electrochemical experiments were performed with acomputer-controlled CHI 660B electrochemical workstation(ChenHua instruments Co. CHI, Shanghai, China), and aplatinum wire as an auxiliary, a saturated calomel electrode(SCE) as a reference and the modified GCE or bare GCE asa working electrode in a conventional three-electrodeelectrochemical cell at room temperature. All potentialsreported in this paper were references to the SCE. Thesolutions were thoroughly deoxygenated for 15 min bybubbling highly purified nitrogen and a nitrogen atmo-sphere was maintained over the solutions. The fieldemission scanning electron microscopy (FE-SEM) imageswere taken using a JEOL JSM-67700F SEM.

Construction of the modified electrode

Prior to the modification, the GCE was polished with emerypaper and 0.05 μm Al2O3 slurry, and then was rinsed withdoubly distilled water, followed by sonicating with 1:1HNO3 solution(Volume ratio of HNO3 and water), acetoneand doubly distilled water for 5 min, respectively. Then theGCE was dried with nitrogen gas, and was activated by 20cyclic sweeping from −0.2 to 1.6 V at 100 mV s−1 in 1 molL−1 H2SO4 aqueous solution until a stable cyclic voltammo-Scheme 1 The chemical structure of ferrocenecarboxamide

114 Microchim Acta (2009) 164:113–118

gram was obtained. A droplet (5 μL) water suspension ofthe MWCNTs (0.5 mg mL−1) was spread on the disksurface of the freshly pretreated electrode using a micro-syringe. Then the electrode was dried in the air forming theMWCNTs modified GCE, denoted as the MWCNTs/GCE.Then the MWCNTs/GCE was immersed into the 3 mmolL−1 HAuCl4 solutions for 3 min [29], after which themodified electrodes were rinsed with copious amounts ofwater and ethanol, dried in a N2 stream and characterizedby FE-SEM, and then NG/MWCNTs/GCE was obtained.After rinsing with water, the NG/MWCNTs/GCE wasimmersed into a PBS (pH=7.0) containing 3 mmol L−1 ofFcAI for 24 h, and then rinsed successively with PBS anddistilled water, and dried with nitrogen gas [24], and thenFcAI/NG/MWCNTs/GCE was successfully fabricated bylinking FcAI to NG through the Au–N bond. Scheme 2shows how a modified electrode can be constructed.

Results and discussion

The characterization of electrode surface

The FE-SEM images of the surfaces of the MWCNTs/GCEand the NG/MWCNTs/GCE are shown in Fig. 1a and b,respectively. From the FE-SEM images, it could be clearlyshown that the NG could be spontaneously formed in andon the matrix of MWCNTs, that is attributed to direct redoxreaction between nanotubes and metal ions [29]. And after

the AuCl4− obtained electron from CNT, it then become Au

nanoparticles and firmly connect with the wall of CNT:

AuCl�4 þ 3e� $ Auþ 4Cl�:

Electrocatalysis of bilirubin modified electrodes

Figure 2 shows the CVs of bilirubin at bare electrodes andmodified electrodes in 0.05 mol L−1 Tris buffer (pH=8.0).The curves (a), (b), (c) and (e) corresponded to theoxidation of 1.0×10−5 mol L−1 bilirubin at the bare GCE,MWCNTs/GCE, NG/MWCNTs/GCE and FcAI/NG/MWCNTs/GCE, respectively, and the curve (d) corre-sponded to the CV of the FcAI/NG/MWCNTs/GCE under0.05 mol L−1 Tris buffer (pH=8.0). There is a good pair ofredox peak of CV curveson FcAI/GNP/MWCNTs/GCE in theblank solution (Fig. 2d) and ΔEp is 0. 07 V, Ipa/Ipa≈1 whichwas caused by the reversible redox process of FcAI and itsuggest that FcAI has been modified onto the electrode. Theanodic peak potential (Epa) for bilirubin was about at 0.45 Vat bare electrodes and modified electrodes (we suggest thatthe chemical equation for the bilirubin oxidation is bilirubin−2e→biliverdin+H+). It could be observed from the figurethat bilirubin exhibits a poor electrochemical response at thebare GCE but better response at the modified electrodes. Thelarger oxidation current may be attributed to the enlargedeffective surface area of the electrodes by the MWCNTs andNG modification. The anodic peak current (Ipa) at the FcAI/

GCE GCE

FcAI( ) MWCNTs HAuCl4

---GNP

GCE GCE

Scheme 2 The construction ofthe modified electrode

Fig. 1 a FE-SEM image of theMWCNTs/GCE; b FE-SEMimage of the NG/MWCNTs/GCE

Microchim Acta (2009) 164:113–118 115

NG/MWCNTs/GCE (curve e) is about 100 times of that atthe bare GCE (curve a), and about 5 times of that at theMWCNTs/GCE (curve b) or at the NG/MWCNTs/GCE(curve c). This increase in the anodic current might beattributed to the electrocatalyzed oxidation of bilirubin,mediated by the Fc+/Fc couple. These indicated that NGand MWCNTs not only had excellent catalytic activity butalso were an excellent link with FcAI through self-assembly,and electron transferred rapidly through FcAI/NG/MWCNTs junction between bilirubin and the underlyingelectrode.

Effect of scan rate

As shown in Fig. 3a, the influence of the scan rate on theoxidation of bilirubin at the FcAI/NG/MWCNTs/GCEelectrode was investigated by CVs. The oxidative peak ofthe modified electrodes in bilirubin solution increased

linearly with the scan rate in the range from 10 to200 mV s−1 with a correlation coefficient of 0.9965(Fig. 3b). The linear regression equation was expressed asIpa(μA)=0.8788+13.046v (V s). The result suggested thatthe electrode reaction of bilirubin was a surface controlledprocess [30] in the range from 10 to 200 mV s−1.

Amperometric response of bilirubin

Figure 4 shows the amperometric response of the FcAI/NG/MWCNTs/GCE on successive addition of 10 μL (0.5 mM)bilirubin to a stirring 5 mL 0.05 Tris buffer (pH=8.0) at theapplied potential of 0.45 V. When the bilirubin was addedinto the buffer solution, the oxidation current rose steeply toreach a stable value. It can be seen that the sensor respondedvery rapidly, producing 95% of the steady-state current within5 s. The steady-state current (Iss) is linearly proportional tobilirubin concentration from 1 to 100 μmol L−1 with acorrelation coefficient of 0.9994. The regression equation isexpressed as Iss=0.1366C+0.0414 (Iss: μA, C: μmol L−1).

-0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7-30

-25

-20

-15

-10

-5

0

5

10

15

20

25

30I/

μA

A

B

D

E

C

E/V(vs.SCE)Fig. 2 Cyclic voltammetries (CVs) of 1.0×10−5 mol L−1 bilirubin onbare GCE (A), MWCNTs/GCE (B), NG/MWCNTs/GCE (C) andFcAI/NG/MWCNTs/GCE (E) in 0.05 mol L−1 Tris buffer (pH=8.0),and the CV (D) of the FcAI/NG/MWCNTs/GCE under 0.05 mol/LTris buffer (pH=8.0). Scan rate=20 mV s−1

-0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7-200

-150

-100

-50

0

50

100

150

I/μA

a

j0.00 0.05 0.10 0.15 0.20 0.250

5

10

15

20

25

30

35

Ipa

/μA

ν/Vs-1

ba

E/V (vs.SCE)

F i g . 3 a CVs o f 1 . 0 ×10−5 mol L−1 bilirubin at theFcAI/NG/MWCNTs/GCE in0.05 mol/L Tris buffer (pH=8.0), scan rates v (V s−1): a 0.01,b 0.02, c 0.03, d 0.04, e 0.06, f0.08, g 0.10, h 0.14, i 0.18, j0.20. b Relationship of the peakcurrent Ipa and the scan rate v(V s−1)

0 100 200 300 400 500 600

0.0

0.5

1.0

1.5

2.0

I/μ

A

Time/sFig. 4 Typical amperometric response of FcAI/NG/MWCNTs/GCE onthe successive addition of 10 μL (0.5 mmol L−1) bilirubin to a stirring5 mL 0.05 Tris buffer (pH=8.0). Applied potential=0.45 V vs. SCE

116 Microchim Acta (2009) 164:113–118

The detection limit (S/N=3) for bilirubin on the electrodemodified with FcAI is estimated to be 1.2×10−7 mol L−1,which could be used as a potential application of themodified electrode to detect bilirubin.

The stability and reproducibility of the FcAI/NG/MWCNTs/GCE

To testify the stability and reproducibility of the FcAI/NG/MWCNTs/GCE, the modified electrodes were stored in0.1 mol L−1 pH 7.0 PBS at 4 °C for 2 weeks after the aboveexperiment, the cyclic voltammetry peak current andpotential for the responses to1.0×10−5 mol L−1 bilirubinwere maintained essentially unchanged. Even if the electro-des were stored for 4 weeks, the peak current response onlyslightly decreased (8.7%) compared with that on the freshfabricated electrodes. These results indicated that themodified electrodes possessed good stability. Bilirubincould be easily adsorbed on the surface of electrodes,which would affect the subsequent measurement. However,renewal of the electrode could be easily accomplished bycycling in PBS for 20 scans from −0.6 to 1.5 V before eachdetermination. The reproducibility of the same GCEelectrode, modified respectively seven times in the samemanner, showed a satisfactory result with the relativestandard deviation (R.S.D.) of 3.5%. Seven modifiedelectrodes were used to estimate the reproducibility of thesensor, and the results revealed it has a satisfied reproduc-ibility with a R.S.D. of 4.3%.

Conclusion

In conclusion, the FcAI/NG/MWCNTs/GCE were success-fully fabricated, characterized and applied in electrocata-lytic oxidation to bilirubin. Owing to excellentelectrochemical catalytic activity of FcAI, NG andMWCNTs, the modified electrode exhibited an excellentelectrochemistry character. The FcAI/NG/MWCNTs/GCEexhibited a highly electrocatalytic activity toward theoxidation of bilirubin and could effectively catalyze theoxidation of B bilirubin. These results showed thatthe method had a wider linear range, low detection limit,fast response, excellent reproducibility and stability. More-over, the fabricating of FcAI/NG/MWCNTs/GCE is simple,rapid, reliable and low-cost. These attractive features provethat this novel electrode is promising to prepare practicalbiosensors.

Acknowledgments The authors gratefully acknowledge the finan-cial support provided by the National Natural Science Foundation(20675001), the Anhui Provincial Natural Science Foundation

(No.050460301), the Science Foundation of Education Office ofAnhui Province (No.2006KJ1198, KJ2007A076, KJ2008B182), theScientific Research Sustentation Program of Anhui Province CollegeYoung Teacher (2005JQ1048ZD), and the Natural Science Foundationof Suzhou College (2007yzk04).

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