Biosensors and Bioelectronics 19 (2003) 227�/232

www.elsevier.com/locate/bios

Direct electrochemistry of horseradish peroxidase bonded on aconducting polymer modified glassy carbon electrode

Young-Tae Kong, Mannan Boopathi, Yoon-Bo Shim *

Department of Chemistry, Pusan National University, Kumjeong-Ku, Pusan 609-735, Republic of Korea

Received 28 May 2002; received in revised form 17 April 2003; accepted 15 May 2003

Abstract

Direct electron transfer process of immobilized horseradish peroxidase (HRP) on a conducting polymer film, and its application

as a biosensor for H2O2, were investigated by using electrochemical methods. The HRP was immobilized by covalent bonding

between amino group of the HRP and carboxylic acid group of 5,2?:5?,2ƒ-terthiophene-3?-carboxylic acid polymer (TCAP) which is

present on a glassy carbon (GC). A pair of redox peaks attributed to the direct redox process of HRP immobilized on the biosensor

electrode were observed at the HRP j TCAP j GC electrode in a 10 mM phosphate buffer solution (pH 7.4). The surface coverage of

the HRP immobilized on TCAP j GC was about 1.2�/10�12 mol cm�2 and the electron transfer rate (ks ) was determined to be 1.03

s�1. The HRP j TCAP j GC electrode acted as a sensor and displayed an excellent specific electrocatalytic response to the reduction

of H2O2 without the aid of an electron transfer mediator. The calibration range of H2O2 was determined from 0.3�/1.5 mM with a

good linear relation.

# 2003 Elsevier B.V. All rights reserved.

Keywords: Amperometric biosensor; Horseradish peroxidase; Direct electrochemistry of HRP; Conducting polymer

1. Introduction

The immobilization of enzymes and diverse biocata-

lysts in conducting polymers are very important for the

development of biosensors (Malinauskas, 1999). A

promising immobilization method, intensively investi-

gated during the last decade, is the entrapment of the

redox proteins or enzymes into hydrogels consisting of a

highly hydrophilic polymer backbone e.g. poly(vinyl

pyridine), poly(vinyl imiazol), poly(acrylic acid), or

poly(allyl amine) (Gregg and Heller, 1990; Degani and

Heller, 1989). Although, related enzyme electrodes have

shown extraordinary sensor properties, the manual

formation of the cross-linked hydrogels remained an

unsolved problem concerning reproducibility and mass

production capability. The physical entrapment is the

simplest way but the catalytic activity of the enzymes is

* Corresponding author. Tel.: �/82-51-510-2244; fax: �/82-51-514-

2430.

E-mail address: ybshim@pusan.ac.kr (Y.-B. Shim).

0956-5663/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S0956-5663(03)00216-1

drastically reduced down to a few percent of the value

observed in the bulk (Calvo and Etchenique, 1996).

In contrast, amperometric biosensors based on the

immobilization of enzymes in an electronically conduct-

ing polymer matrix are of great interest. Advantages of

the electrochemical formation of conducing-polymer

films are: (1) simple preparation procedure; (2) minia-

turization; and (3) the precise localization of the

polymer thickness and enzyme. Furthermore, it takes

place exclusively on the electrode surface.

Some authors (Dobey et al., 1999; Wallace et al.,

1999; Schuhmann, 1995; Cosneir, 1999; Yamauchi et al.,

1999; Schuhmann, 2002) have reported that the glucose

oxidase-immobilized glucose sensor prepared by electro-

polymerization of pyrrole derivatives that have carboxyl

and hyrocarboxyl groups, affords higher electrode

stability and reproducibility compared with that of

unsubstituted pyrrole.

Horseradish peroxidase (HRP) is an enzyme, which

catalyzes the hydrogen peroxide-dependent-one-electron

oxidation of a wide variety of substrate (Hewson and

Hager, 1979). Also HRP has long been a representative

system for investigating especially for their biological

Scheme 1. Schematic representation of the present study.

Y.-T. Kong et al. / Biosensors and Bioelectronics 19 (2003) 227�/232228

behaviors for catalyzing oxidation of substrates by

hydrogen peroxide (Bosshard et al., 1991). Electroche-

mical techniques have been proven to be a valuable tool

for the study of heme proteins and applied to construct

an enzyme electrode. Through the occurrence of direct

electron transfer between adsorbed HRP and the

electrode has been demonstrated (Yaropolov et al.,

1978). In some earlier reports, covalent modificationof HRP and other enzymes were carried out on the

electrode surface which are having a self assembled

monolayer, in one case redox signal for the enzymes

were observed (Lotzbeyer et al., 1996), however, in an

another case they are not able to get the electrochemical

redox signals for HRP (Lotzbeyer et al., 1997).

To our knowledge, so far there is no report on direct

electrochemistry of HRP using a novel conductingpolymer (5,2?:5?,2ƒ-terthiophene-3?-carboxylic acid poly-

mer (TCAP)) (Lee et al., 2002). Thus, in this study, we

report the direct electron transfer properties of the HRP

using the TCAP modified on a glassy carbon (GC)

electrode using electropolymerization method for the

polymer coating process. The HRP enzyme was cova-

lently bonded to the carboxylic acid group of the TCAP

using a catalyst. The effect of HRP immobilization timefor the covalent bonding and the effect of pH after the

modification of HRP were optimized. The process of

direct electrochemistry attained based on the present

HRP modified electrode was utilized for the ampero-

metric measurement of H2O2 in the absence of a

mediator.

2. Experimental

2.1. Reagents

Peroxidase from horseradish (EC.1.11.1.7, 180 U

mg�1) was purchased from Toyobo, Japan and was

used as received. A terthiophene (5,2?:5?,2ƒ-terthio-

phene-3?-carboxylic acid) monomer bearing a carboxyl

group, was synthesized as described previously (Lee et

al., 2002). 1-Ethyl-3-(3-dimethylamino-propyl)carbodii-

mide (EDC) was purchased from the sigma. All other

chemicals were of analytical grade and were used

without further purification. All experimental solutions

were prepared in doubly distilled water obtained from aMilli-Q water system.

2.2. Preparation of HRP-immobilized electrode

(HRP j TCAP j GC)

The HRP immobilization was achieved by forming

peptide bonds between the amine groups of the HRP

and the carboxylic acid groups of the TCAP present on

the GC. The thoroughly washed GC electrode coated

with TCAP bearing carboxylic acid groups was used to

immobilize the HRP, as shown in Scheme 1. Formodification, the HRP was immobilized on the TCAP

coated GC electrode (area, 0.07 cm2). The polymer film

was formed on the GC using a 0.1 M Bt4NClO4/CH2Cl2solution containing a 1.0 mM monomer TCAP by

potential cycling five times from 0.0 to �/1.5 V vs

Ag j AgCl (Fig. 1) (Lee et al., 2002).

The polymer-coated electrode was immersed in to a

10 mM phosphate buffer solution (pH 7.4) containing5.0 mM EDC for 12 h. The EDC-attached electrode was

washed with a buffer solution and subsequently incu-

bated in a 100 mM HRP prepared in a phosphate buffer

solution for 48 h at 4 8C. By this procedure, the HRP

was immobilized on the terthiophene film through the

formation of covalent bonds with carboxyl groups on

the polymer.

Prior to coating the TCAP on the GC by theelectrochemical technique, we pretreated the GC as

follows: the GC was polished using an alumina slurry

(0.05 mm) on a polishing cloth and washed with

Fig. 1. Electropolymerization of 1 mM 5,2?:5?,2ƒ-terthiophene-

3?carboxylic acid (TTCA) monomer in CH2Cl2 containing 0.1 M

TBAP. Potential cycling between 0.0 and 1.5 V vs Ag j AgCl at a GC

electrode at scan rate: 100 mV s�1.

Fig. 2. Cyclic voltammograms of: (1) polymer coated (TCAP j GC)

electrode; and (2) HRP j TCAP j GC modified electrode in phosphate

buffer solution, pH 7.4 at scan rate 100 mV s�1.

Y.-T. Kong et al. / Biosensors and Bioelectronics 19 (2003) 227�/232 229

deionized water followed by ultrasonication. Afterpolishing, the electrochemical pretreatment was per-

formed by a potential step applying �/0.85 V for 5 min

and �/1.4 V for 1 min in 0.1 M H2SO4. Reproducible

electrochemical responses were not obtained without

using EDC, confirming the formation of the binding

between amino groups on HRP surface and carboxylic

groups on the conducting polymer. Amino groups on

the HRP surface responsible for the binding are notspecified now.

2.3. Electrochemical measurements

Electrochemical measurements were performed with athree-electrode system. The HRP j TCAP j GC elec-

trode, an Ag j AgCl (saturated KCl) electrode, and a

platinum wire were used as the working, the reference,

and the auxiliary electrode, respectively. An EG&G

PAR 273A potentiostat/galvanostat was used for vol-

tammetric experiments. Amperometric experiments

were carried out in a stirred system by applying a

potential of �/200 mV to the working electrode.Aliquots of H2O2 standard solution were successively

added to the solution. Current-time data was recorded

after a steady-state current had been achieved. All

experimental solutions were deaerated with nitrogen

gas for at least 10 min and maintained under a nitrogen

atmosphere during voltammetric measurements.

3. Results and discussion

Fig. 2 shows the cyclic voltammograms of the

HRP j TCAP j GC (curve 2) and mere TCAP j GC

(curve 1) electrodes in a phosphate buffer solution ofpH 7.4 at 100 mV s�1 of the scan rate, respectively. As

shown in CVs (Fig. 2), only the HRP j TCAP j GC

electrode displays a pair of redox peaks at 90 mV for Epa

and �/190 mV for Epc. The cathodic and anodic peaks

are symmetric and of similar magnitude, with the ipa/ipc

ratio was about unity. These peaks result from the redox

process of HRP immobilized on conducting polymer.

The favored orientation of the HRP molecules and the

conducting channels of polymer can achieve the direct

electron transfer reaction between the prosthetic groups

of HRP and the conducing polymer coated on the

electrode surface.

The spectrometric method (Imabayashi et al., 2001)

described previously was employed to estimate the

apparent amount of HRP immobilized on the conduct-

ing polymer modified electrode. The HRP j TCAP j GC

electrode was immersed in a fixed volume of the solution

containing 0.3 wt.% of pyrogallol and 10 mM H2O2.

After 5 min, the absorption due to the generation of

oxidized pyrogallol was recorded. This absorption can

be correlated to the amount of active HRP from the

calibration plot and the absorbance resulting from the

HRP immobilized on the electrode. The apparent

amount of immobilized active HRP was estimated to

be of the order of 1.3�/10�12 mol cm�2. This value

corresponds to 1/10�/1/40 of the surface coverage for the

HRP on the polycrystalline gold disk electrode (Li and

Dong, 1997). The coverage did not significantly increase

when the reaction time for the HRP immobilization was

prolonged more than 48 h (not shown).Fig. 3 displays a pair of redox peaks for the direct

electron transfer behavior of immobilized HRP at

various scan rates. The ratio of cathodic to anodic

peak currents is nearly unity. Peak currents vary linearly

with the scan rate, as shown in the inset of Fig. 3,

indicating the electrode process is not diffusion-con-

trolled, as expected for an immobilized system. The

cathodic and anodic peak potentials shift very slightly in

opposite directions with a change of scan rate. The

peak-to-peak separation is 280 mV at a scan rate of 100

mV s�1, which is similar to those obtained by other

systems (Yi et al., 2000). The larger Epa/Epc is probably

Fig. 3. Cyclic voltammograms of HRP j TCAP j GC electrode in

phosphate buffer solution, pH 7.4 at various scan rates. A plot of

peak current vs scan rate is shown in inset.

Fig. 4. Effect of pH on the anodic peak current of the electrochemical

response of HRP j TCAP j GC electrode.

Fig. 5. Typical amperometric responses at the HRP j TCAP j GC

electrode to successive additions of H2O2 at �/200 mV vs Ag j AgCl.

Y.-T. Kong et al. / Biosensors and Bioelectronics 19 (2003) 227�/232230

ascribable to the immobilized HRP molecules in various

orientations (Li and Dong, 1997). We calculated the

electron transfer rate constant (ks) as 1.03 s�1 using the

data for 50 mV s�1 from Fig. 3 by adopting an earlier

reported method (Laviron, 1979). The ks value obtained

in this study is very high when compared to the previous

studies in which they did not use any conducting

polymer (Tatsuma et al., 1998; Ruzgas et al., 1995) on

the carbon surface, from the ks value we conclude that

in our study the use of TCAP enhanced the electron

transfer rate. However, our ks value is found to be less

when compared to a very recent report (Ferapontova et

al., 2002), because their system is quite different from

ours and also they used recombinant HRP instead of

using native HRP what we used. It is noteworthy that no

corresponding peak is observable with HRP at either a

bare GC or a TCAP j GC electrode in the same

potential range (not shown). Therefore, this pair of

peaks comes from the redox reaction of the electroactive

sites of HRP on the conducting polymer modified

electrode. Considering the fact that HRP alone exhibits

no observable peak at the TCAP j GC electrode surface,

thus, the electron transfer process is considerably

enhanced by the conducting polymer due to the covalent

attachment of HRP on it.

The changes in the anodic peak height were examined

as a function of the pH on the electrochemical response

of the HRP j TCAP j GC electrode in the pH range of

6.5�/8.5. As shown in Fig. 4, maximum peak height was

achieved around pH 7.5. This optimum pH is good

agreement with that reported for an enzyme (Reiter et

al., 2001). Therefore, a pH value of 7.4 was selected for

this study due to the biological significance of HRP.

Since we obtained direct electron transfer process with

HRP j TCAP j GC electrode. We decided to use this

modified electrode for catalytic purposes. The immobi-

lized HRP undertakes a direct electron transfer reaction

and up on testing it exhibits an excellent electrocatalytic

response to the reduction of H2O2 in a phosphate buffer

solution, pH 7.4. Therefore, we applied the

HRP j TCAP j GC electrode for determination of

H2O2. From the dependence of the peak current on

the applied voltage at the HRP j TCAP j GC electrode,

we chose a working applied potential of �/200 mV for

the amperometric determination of H2O2, where the risk

for interference from reactions of oxygen and other

electroactive substances in the solution was minimized.

The catalytic mechanism of immobilized HRP to H2O2

reduction has already been proposed (Ferri et al., 1998;

Foulds and Lowe, 1988).

Fig. 5 shows the typical amperometric responses by

the HRP j TCAP j GC electrode with successive addi-

tions of 0.2 mM H2O2. The steady-state current values

increased linearly with the concentration of H2O2 up to

1.5 mM with a correlation coefficient of 0.995 and

deviated downward from the linear relationship in the

higher concentration region due to the HRP reaction

kinetics (Fig. 6). The detection limit determined was 0.2

mM at a signal-to-noise ratio of three. The reproduci-

Fig. 6. Calibration plot for H2O2 obtained with HRP j TCAP j GC

electrode at a potential of �/200 mV vs Ag j AgCl. Inset is the linear

range of calibration plot.

Y.-T. Kong et al. / Biosensors and Bioelectronics 19 (2003) 227�/232 231

bility expressed in terms of relative standard deviation

was 2.2% (n�/5) for a concentration of 5�/10�4 M

H2O2. The sensor showed a fast response time (t95%) of

10 s.

The stability of the HRP j TCAP j GC electrode was

studied in the linear range of H2O2. When the H2O2

sensor was stored in a phosphate buffer solution, pH 7.4

for 3 weeks at 4 8C, the sensor retained over 90%

response of its initial sensitivity to the reduction of

H2O2. The deterioration of the sensor response beyond 3

weeks period may be due to the deactivation of

immobilized HRP. When the concentration of H2O2

was higher than 4.0 mM, denaturation of HRP

occurred, and the HRP was probably irreversibly

transformed into its higher oxidized, inactive form.

Thus, high concentration of H2O2 should be avoided

in order to maintain the bioactivity of the HRP over a

longer period of time.

4. Conclusion

The present paper demonstrates that HRP was

covalently immobilized on a conducting polymer coated

GC electrode. This method is advantageous when

compared to other immobilization procedures because

of very short time is enough for electropolymerization.

The covalent attachment of HRP on the TCAP matrix

rendered direct redox property to the HRP. The

polymer also enhanced the electron transfer rate (ks )

when compared to other studies in which no polymer

was used on carbon electrodes. The modified electrode

system acts as an efficient and stable amperometric

biosensor. In absence of mediator, the direct electro-

chemistry of HRP offers an opportunity to build up a

biosensor for H2O2. The calibration plot shows a linear

range between the H2O2 concentrations from 0.3 to 1.5

mM with a detection limit of 0.2 mM.

Acknowledgements

The authors greatly appreciated the financial support

by the Korean Research Foundation Grant (KRF 00-015-DP0238), S. Korea.

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