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Electrochemistry Communications 8 (2006) 1572–1576

Direct electron transfer of redox proteins on a Nafion-cysteinemodified gold electrode

Jun Hong, Hedayatollah Ghourchian *, Ali Akbar Moosavi–Movahedi

Institute of Biochemistry and Biophysics, University of Tehran, Enquelab Avenue, P.O. Box 13145-1384, Tehran, Iran

Received 7 June 2006; received in revised form 9 July 2006; accepted 11 July 2006Available online 17 August 2006

Abstract

A novel Nafion-cysteine functional membrane was constructed. Rapid and direct electron transfer of horseradish peroxidase, Euphor-bia latex amine oxidase, superoxide dismutase and cytochrome c was carried out on the functional membrane modified gold electrodewith good stability and repeatability. The immobilized protein modified electrodes showed quasi-reversible electrochemical redox behav-iors with formal potentials of 60, 35, 66 and 38 mV (vs. Ag/AgCl) in 20 mM potassium phosphate buffer solution, pH 7.0 at 25 �C. Thecathodic transfer coefficients were 0.42, 0.42, 0.45, 0.44 and electron transfer rate constants were evaluated to be 1.2, 1.6, 1.0 and 0.6 s�1.� 2006 Elsevier B.V. All rights reserved.

Keywords: Direct electron transfer; Horseradish peroxidase; Euphorbia latex amine oxidase; Superoxide dismutase; Cytochrome c; Nafion

1. Introduction

The study of direct electron transfer in proteins andenzymes represents a basic feature for the application ofbiocatalysts in chemical sensors and other electrochemicaldevices [1,2]. Great progress in this field has shown thatthe modified films on electrodes may provide a favorablemicroenvironment for proteins to exchange electronsdirectly with underlying electrodes, and thus afford a newopportunity for the detailed study of enzyme electrochem-istry [3–5]. Successful approaches have included cast filmsof proteins with insoluble surfactants [6–8], biologicalorganic substances [9–11], inorganic membranes [12–15],and films of proteins and polyions grown layer-by-layer[16–18]. In addition, protein- or enzyme-containing thinfilms modified on electrode surfaces have potential applica-bility in manufacturing biosensors, biomedical devices, andenzymatic bioreactors [19,20]. Achieving direct electronexchange between redox proteins or enzymes and elec-trodes simplifies these devices by removing the requirement

1388-2481/$ - see front matter � 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.elecom.2006.07.011

* Corresponding author. Fax: +98 21 66404680.E-mail address: hadi@ibb.ut.ac.ir (H. Ghourchian).

of chemical mediators, and thus has a great significance inpreparing the third generation biosensors [21].

As reported previously [22,23], an ideal promoter usedfor direct electron transfer of proteins and enzymes shouldprovide a suitable interface between the proteins and theelectrode surface to partly or completely eliminate thedenaturation of bio-macromolecules on the electrode sur-face and to promote the electron transfer between proteinsand the electrode.

Nafion is a perfluorinated anionic polyelectrolyte [24].The increasing popularity of Nafion for the fabrication ofredox polymer-modified electrodes in recent years arisesfrom its ease of use, good electrical conductivity and thehigh partition coefficients of many redox compounds inNafion. In addition, Nafion film has high chemical stabil-ity, good biocompatibility and the ability to resist interfer-ences from anions and biological macromolecules [25–27].

Cysteine is an amino acid that plays important roles inbiological systems. Tian et al. used this amino acid as anelectron transfer promoter to construct a self-assembledmonolayer (SAM) and applied that monolayer to studydirect electron transfer between a gold electrode and super-oxide dismutase [22,28–30]. The SAM, thus prepared onthe electrode surface, was very thin, weak and labile.

J. Hong et al. / Electrochemistry Communications 8 (2006) 1572–1576 1573

Furthermore, a low molecular weight (MW) promoter likecysteine may leach out of the electrode and therefore can-not offer a suitable biocompatible microenvironment forproteins or bio-macromolecules, which may lead to signif-icant signal loss and affect the stability of the biosensor. Inorder to overcome these shortcomings, we have developeda new type of functional membrane composed of cysteineand Nafion. The functional membrane could provide suit-able sites for adsorbing bio-molecules, thus promoting theelectron transfer of proteins on the gold electrode. In thisreport, a Nafion-cysteine membrane was used not only topromote the electron transfer rate but also to offer a bio-compatible microenvironment on the electrode surface.The direct electron transfer of redox proteins, includinghorseradish peroxidase (HRP), euphorbia latex amine oxi-dase (ELAO), superoxide dismutase (SOD) and horse heartcytochrome c (Cyt c), was studied.

2. Experimental

2.1. Materials

HRP (EC 1.11.1.7), SOD (EC 1.15.1.1), catalase (EC1.11.1.6), hemin (C34H32ClFeN4O4), and Nafion� (5 wt %ethanol solution) were purchased from Sigma. ELAOwas purified as previously described [31–33]. Cyt c andother reagents (analytical grade) were purchased fromMerck. The aqueous cysteine solution was freshly preparedand deoxygenated by bubbling pure N2 gas for at least30 min prior to use. The solutions were prepared in deion-ized double distilled water (18 MX cm, Barnstead, Dubu-que, USA).

2.2. Preparation of Nafion-cysteine-modified gold electrode

The preparation of the gold electrode was similar to pre-viously described procedures [22,28–30]. Briefly, the goldelectrode was mechanically polished twice with alumina(particle sizes 10 and 0.3 lm) to a mirror finish. Then, itwas ultrasonically treated in water for 10 min. Thereafter,it was treated electrochemically in 0.2 M sulfuric acid,cycling between �0.2 and +1.5 V (vs. Ag/AgCl) at a sweeprate of 0.1 V s�1 until the appearance of a clean gold elec-trode was obtained. Finally the electrode was washed withdeionized double distilled water.

Nafion solution (2 ll, 5%) was dropped onto the sur-face of freshly prepared gold electrode and dried at roomtemperature for 20 min. Then the electrode was dippedinto a freshly prepared cysteine solution (10 mM) for10 min and carefully washed with deionized double dis-tilled water.

The protein (ELAO, HRP, SOD or Cyt c) was immobi-lized by dropping 2 ll of 10 mg/ml of protein solution onthe Nafion-cysteine modified gold electrode and dried forabout 30 min at room temperature. Then the electrodewas gently washed with deionized double distilled waterand stored at 4 �C when not in use.

2.3. Apparatus and measurements

Electrochemical measurements were carried out with apotentiostat/galvanostat (Model 263A, EG&G, USA)using a single-compartment voltammetric cell equippedwith a platinum rod auxiliary electrode, an Ag/AgCl refer-ence electrode (Metrohm) and a gold working electrodewith a disk diameter of 1 mm (Azar Electrode, Iran). Theexperimental solutions were deaerated using highly purenitrogen for 30 min and a nitrogen atmosphere was keptover the solutions during measurements. All the electro-chemical measurements were carried out in 20 mM phos-phate buffer solution, pH 7.0 at 25 ± 1 �C.

The surface morphology of the Nafion-cysteine mem-brane on the gold-coated glass substrate was studied byscanning electron microscopy (SEM) (DSM 960 A, Zeiss,Germany). The membrane was prepared using the samemethod as that of the Nafion-cysteine gold electrode.

3. Results and discussion

Fig. 1 represents the electron transfer properties of theimmobilized redox proteins (ELAO, HRP, SOD or Cytc) on the Nafion-cysteine modified gold electrode at differ-ent sweep rates. A pair of well-defined redox peaks wasobserved which could be ascribed to the direct electrontransfer of protein at the modified electrode at each sweeprate. The direct electron transfer of the redox protein at thebare gold electrode is very slow so that the redox peak usu-ally cannot be observed [22,34]. Furthermore, no redoxpeaks were observed for redox proteins at the Nafion-mod-ified gold electrode (without cysteine) nor for the Nafion-cysteine modified gold electrode (without protein – resultsnot shown). However, the Nafion-cysteine functional mem-brane significantly promotes the electron-transfer rates ofproteins (as depicted in Fig. 1). According to some reports,cysteine bridges between the protein and the electrodefunction as promoters for the electron transfer process[22,28,29]. Nafion also improves the stability of the pro-teins and offers a biocompatible microenvironment on theelectrode surface [25–27].

The electrochemical and kinetic parameters of the pro-teins immobilized on the functional membrane are givenin Table 1. The rate constant of the electrode reaction(ks) and cathodic transfer coefficient (ac) for the redoxreaction of the proteins confined on the modified goldelectrode were evaluated based on Laviron’s Equation[35,36].

Based on the observed cyclic voltammograms (CVs) ofthe proteins (Fig. 1), the peak currents increased withincreasing sweep rate (v) and they were proportional to v

(not v1/2) in the range of 0.02–1.0 V s�1. The integrationof reduction peaks at different sweep rates gave a nearlyconstant charge (Q) value. These are characteristics of dif-fusionless, thin-layer electrochemical behavior [37].

As represented in Table 1, the proteins have nearly thesame cathodic transfer coefficients (0.44 ± 0.02) in the

-6

-4

-2

0

2

4

6

-0.6 -0.4 -0.2 0 0.2 0.4 0.6

E/V vs. Ag/AgCl

I /µ

A

-5

-4

-3

-2

-1

0

1

2

3

4

-0.6 -0.4 -0.2 0 0.2 0.4

E/V vs. Ag/AgCl

I /µ

A

-6

-4

-2

0

2

4

6

-0.6 -0.4 -0.2 0 0.2 0.4

E/V vs. Ag/AgCl

I /µ

A

-5

-4

-3

-2

-1

0

1

2

3

4

-0.6 -0.4 -0.2 0 0.2 0.4

E/V vs. Ag/AgCl

I /µA

a b

c d

Fig. 1. Cyclic voltammograms of (a) Nafion-cysteine-HRP, (b) Nafion-cysteine-ELAO, (c) Nafion-cysteine-SOD and (d) Nafion-cysteine-Cyt c modifiedgold electrode in 20 mM potassium phosphate buffer solution (pH 7.0). Sweep rates (from inner to outer) for (b) and (c): 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0 V s�1 and for (a) and (d): 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 V s�1.

1574 J. Hong et al. / Electrochemistry Communications 8 (2006) 1572–1576

redox reaction. In addition, the measured formal potentialof SOD was 65 ± 5 mV vs. Ag/AgCl (0.26 V vs. NHE),which is very close to the value obtained on the cysteine-modified Au electrode reported by Tian et al. [22,28,29].The similarity of these two results seems to be due to pres-ence of cysteine, which acts as the protein electron transferpromoter at gold electrode surface. However, the value ofthe formal potential of SOD (0.26 V vs. NHE) is slightlysmaller than the value reported by other researchers:0.30 V vs. NHE, obtained at a cysteine-modified gold wireelectrode, in phosphate buffer solution (pH 7.0) [38]; and0.32 V vs. NHE, obtained at a gold electrode, in 0.1 MNaClO4 solution (pH 7.2), containing 2 · 10�4 M 1,2-bis(4-pyridyl)ethane [39]. This variation is probably due tothe differences in experimental conditions. Furthermore,the formal potential estimated to be 38 ± 5 mV vs. Ag/AgCl (0.235 V vs. NHE) for the Nafion-cysteine-Cyt c

modified gold electrode is consistent with previous reportsof 0.215 V vs. NHE [40] and 0.234 V vs. NHE [41]. More-over, the value of the formal potential of HRP (0.257 V vs.NHE) is about 0.2 V smaller than that estimated from lit-

erature (about 0.451 V vs. NHE), which obtained at a goldcolloid/cysteine/Nafion modified platinum disk electrodein 5 mM FeðCNÞ�4=�3

6 solution [42]. These evidences indi-cate that the Nafion-cysteine functional membrane actsas a sufficient promoter to carry out direct electron transferbetween redox proteins and gold electrode.

It seems that combination of Nafion and cysteine pro-vides suitable sites for adsorbing bio-molecules. Tianet al. studied the direct electron transfer of SOD at a goldelectrode with a cysteine SAM in phosphate buffer solutioncontaining SOD, and they suggested that the SOD mightbe permanently confined to the electrode surface via theSAM of promoters through various means, such as electro-static and hydrophobic interactions and hydrogen bonding[22]. However, understanding the Nafion-cysteine func-tional membrane is much more complicated. We expectthat, while Nafion-cysteine functional membrane acts asthe protein electron transfer promoter at gold electrodesurface, the Nafion offers a biocompatible microenviron-ment to confine bio-macromolecules in its ionic clusterregion (30–50 nm), based on a stylized, semi-empirical view

Tab

le1

Ele

ctro

chem

ical

par

amet

ers

of

red

ox

pro

tein

so

nN

afio

n-c

yste

ine

fun

ctio

nal

mem

bra

ne

mo

difi

edgo

ldel

ectr

od

e

Pro

tein

sM

ole

cula

rw

eigh

t(M

W)

Ele

ctro

de

reac

tio

nE

00

(mV

vs.

Ag/

AgC

l)E

00

(Vvs

.N

HE

)b

yli

tera

ture

sD

Ep

(mV

)a c

ks

(s�

1)

Ho

rse

hea

rtcy

toch

rom

ec

12,3

80C

ytc½h

emeð

Fe3þÞ�þ

e�

()

Cyt

c½h

emeð

Fe2þÞ�

38±

5(0

.235

Vvs

.N

HE

)0.

215

[40]

388

±5

0.44

±0.

020.

6±

0.1

0.23

4[4

1]B

ovi

ne

eryt

hro

cyte

sup

ero

xid

ed

ism

uta

se32

,500

SO

DðC

u2þÞþ

e�

()

SO

DðC

uþÞ

66±

5(0

.263

Vvs

.N

HE

)0.

30[3

8]29

6±

50.

45±

0.02

1.0

±0.

10.

32[3

9]0.

26[2

2,28

,29]

Ho

rser

adis

hp

ero

xid

ase

44,0

00H

RP½h

emeð

Fe3þÞ�þ

e�

()

HR

P½h

emeð

Fe2þÞ�

60±

5(0

.257

Vvs

.N

HE

)0.

451

[42]

256

±5

0.42

±0.

021.

2±

0.1

Eu

ph

orb

iala

tex

amin

eo

xid

ase

140,

000

EL

AOðC

u2þÞþ

e�

()

EL

AOðC

uþÞ

35±

5(0

.232

Vvs

.N

HE

)N

ore

po

rts

308

±5

0.42

±0.

021.

6±

0.1

E00

and

DE

pw

ere

esti

mat

edasðE

a pþ

Ec pÞ=

2an

dðE

a p�

Ec pÞi

nth

eC

Vs

ob

tain

edw

ith

the

mo

difi

edel

ectr

od

esin

20m

M,

pH

7.0

po

tass

ium

ph

osp

hat

eb

uff

erso

luti

on

at10

0m

V/s

.E

a pan

dE

c pw

ere

ano

dic

and

cath

od

icp

eak

po

ten

tial

so

fth

ep

rote

ins.

J. Hong et al. / Electrochemistry Communications 8 (2006) 1572–1576 1575

of a polar/nonpolar microphase separation in a hydratedionomer [43–51].

Moreover, the thickness of the membrane may alsogreatly affect the electron transfer process. The thinnermembrane was not stable and the thicker one would leadto a weaker redox response or no redox response at all(results not shown). Cross-section SEM image of the Naf-ion-cysteine membrane on the gold coated glass substrateshowed that satisfactory results were obtained from amembrane approximately 150 nm in thickness. Also topview of the SEM image of the membrane showed lots ofmeshy protuberances and hollow structures (image notshown), which seems to result in effective adsorption ofthe protein molecules [52].

The data in Table 1 led us to further conclude that theNafion-cysteine membrane efficiently adsorbs only thoseproteins within a limited range of molecular size. It canbe seen that the electron transfer rate constant (ks)increases with increasing protein molecular weight, rangingfrom 12,400 to 144,000. The greater the molecular size ofprotein the more area available to transfer electrons. More-over, the CVs of the proteins in Fig. 1 and the separationbetween the anodic and cathodic peak potentials (DEp) inTable 1 indicate that the redox peaks of HRP and SODare sharper than those of Cyt c or ELAO, suggesting thatHRP and SOD are more suitable to carry out electrontransfer under the same experimental conditions. Verylow and high molecular weight bio-molecules were alsoexamined with this system. Hemin and catalase, withmolecular weights of 652 and 250,000, respectively, showedalmost no redox response (results not shown). There maybe two different views to explain this evidence regardingthe structure and size of Nafion and cysteine moleculesand their function at microcircumstances. The small mole-cule like hemin may leach out of the membrane and cannotproduce a stable redox response. Moreover, no redox peakwas observed not only for immobilized catalase on Nafion-cysteine-gold electrode but also for cysteine modified goldelectrode in catalase solution or bare gold electrode in cat-alase solution containing cysteine (unpublished results). Itis assumed that large molecules are not able to diffuse intothe membrane net during the immobilization process andthe length of cysteine would not be sufficient to reach theelectro-active site of the large molecules. However, thishypothesis requires further investigation.

4. Conclusions

The direct electron transfer of a redox protein at a baregold electrode is too slow to observe the redox peak. TheNafion-cysteine functional membrane significantly pro-motes the electron-transfer rate of proteins. We believe thatwhile Nafion-cysteine acts as a promoter between the pro-tein molecules and the gold electrode, a biocompatiblemicroenvironment to confine bio-macromolecules is offeredin the ionic cluster region of Nafion. The similarity of theredox reaction cathodic transfer coefficients of all proteins

1576 J. Hong et al. / Electrochemistry Communications 8 (2006) 1572–1576

tested (HRP, ELAO, SOD and Cyt c) seems to be due tocysteine and its special effect. The Nafion-cysteine mem-brane was efficient only with proteins within a limitedrange of molecular size. While the redox peaks of HRPand SOD (medium-sized molecules) were sharper thanCyt c or ELAO, proteins with very low/high molecularweight showed almost no redox response. Thus, HRPand SOD appear to be more suitable proteins for electrontransfer than Cyt c or ELAO. In addition, the proportion-ality of peak currents to sweep rate and the constancy ofintegration of reduction peaks at different scan rates repre-sent the characteristics of diffusion-less, thin-layer electro-chemical behavior.

Acknowledgements

The authors thank Mr. Mojtaba Amani, Mr. Saeed Re-zaei-Zarchi, Mr. Ahmad Moulaei Rad and Dr. ShahinAhmadian (Institute of Biochemistry and Biophysics, Uni-versity of Tehran, Iran), Dr. Nuroozi Parviz and S.M.H.Hashemi (Faculty of Science, University of Tehran, Iran)and Dr. Giovanni Floris (Department of Sciences Appliedto Biosystems, University of Cagliari, Italy) for their kindcollaboration. The financial support provided by the Re-search Council of the University of Tehran and Iran Na-tional Science Foundation (INSF) are gratefullyacknowledged.

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