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Synthetic Metals 161 (2011) 440–444

Contents lists available at ScienceDirect

Synthetic Metals

journa l homepage: www.e lsev ier .com/ locate /synmet

novel amperometric galactose biosensor based on galactosexidase-poly(N-glycidylpyrrole-co-pyrrole)

ehmet Senela,b,∗, Ibrahim Bozgeyikc, Emre Cevikb,d, M. Fatih Abasıyanıkb,d

Department of Chemistry, Faculty of Arts and Sciences, Fatih University, B.Cekmece, Istanbul 34500, TurkeyBiotechnology Research Laboratory, Bionanotechnology Research Center, Fatih University, B.Cekmece, Istanbul 34500, TurkeyDepartment of Biology, Faculty of Arts and Sciences, Fatih University, B.Cekmece, Istanbul 34500, TurkeyDepartment of Genetics and Bioengineering, Faculty of Engineering, Fatih University, B.Cekmece, Istanbul 34500, Turkey

r t i c l e i n f o

rticle history:eceived 25 October 2010eceived in revised form4 December 2010

a b s t r a c t

A novel amperometric galactose biosensor was constructed by immobilization of galactose oxidase(GAox) onto poly(N-glycidylpyrrole-co-pyrrole) film. GAox enzyme was immobilized onto the electro-chemically prepared novel conducting polymer film by direct one-step covalent attachment withoutusing any coupling agents. The biosensor surface was characterized by FT-IR spectroscopy and atomic

ccepted 21 December 2010vailable online 17 January 2011

eywords:iosensoralactose oxidase

force microscopy (AFM). Amperometric response was measured as a function of concentration of galac-tose, at fixed potential of +0.7 V vs. Ag/AgCl in a phosphate buffered saline (pH 7.5). The mediatedhydrogen peroxide biosensor had a fast response of less than 5 s with linear range 2–16 mM. The sensi-tivity of the biosensor for galactose was 1.75 �A/mM. The factors influencing on the performance of theresulting biosensor were studied in detail.

yrrolemmobilization

. Introduction

Conducting polymer interfaces are particularly suitable forocalizing biomolecules onto surfaces [1]. Electrochemically poly-

erized conducting polymers has been studied extensively forhe construction of biosensors, because of the (1) direct and easyeposition on the electrode surface, (2) control of thickness, (3)edox conductivity of and polyelectrolyte characteristics of poly-er useful for sensor application [2]. Polypyrrole fulfills the above

equirements together with having the characteristics of easy oxi-ation, high chemical stability, low cost of monomer [3].

Determination of galactose is important in food science, humanutrition, medicine and fermentation industry. Different tech-iques have been reported for the determination of galactose4–6]. Which are not effective methods due to the colour, tur-idity or particulates. A combination of the immobilized enzymend electrochemical method provides a fast and reliable method

o study the enzyme-catalyzed reaction kinetics and determinehe substrate concentration. Galactose oxidase is the most com-

only used enzyme for galactose determinations [4,7,8]. Variousmperometric biosensors containing the immobilized galactose

∗ Corresponding author at: Fatih University, Department of Chemistry, Buyukcek-ece Kampusu, 34500 B.Cekmece, Istanbul, Turkey. Tel.: +90 2128663300x2067;

ax: +90 2128663402.E-mail address: msenel@fatih.edu.tr (M. Senel).

379-6779/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.synthmet.2010.12.025

© 2010 Elsevier B.V. All rights reserved.

oxidase have been reported to use for the galactose detection[9–11].

In our previous study galactose oxidase was immobilized ontothe ferrocene containing polymeric mediator for determination ofgalactose [12]. Here we report fabrication of a galactose biosensorbased on direct covalent attachment of galactose oxidase on a novelconducting polymer film. Kinetic parameters and the factors influ-encing the performance of the resulting biosensor were studied indetail.

2. Experimental

2.1. Materials and apparatus

Glucose oxidase (GOx) (EC 1.1.3.4) was obtained from Sigma.Pyrrole (Py) and epicholrohydrin (ECH) were obtained from Aldrich.Other chemicals and solvents were of guaranteed reagent- or ana-lytical grade and were used without further purification.

The FTIR-ATR spectra (4000–400 cm−1) were recorded with aBruker spectrometer. NMR spectra were recorded in CDCl3 usinga Bruker 400 MHz spectrometer. The morphology of the modified

gold surfaces was imaged by Atomic Force Microscopy (AFM, Parksystems XE-100E). Imaging was carried out at ambient temperaturein noncontact mode.

Electrochemical polymerizations and measurements were per-formed using a CHI Model 842B electrochemical analyzer. A small

M. Senel et al. / Synthetic Metals 161 (2011) 440–444 441

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Fig. 1. Preparation of poly(N-glycidylpyrrole-co-pyrrole

old working electrode (1 cm2 diameter), a platinum counterlectrode (1 cm2 diameter), an Ag/AgCl-saturated KCl referencelectrode, and a conventional three-electrode electrochemical cellsed were purchased from CH Instruments.

.2. Synthesis of N-glycidylpyrrole monomer

N-glycidylpyrrole monomer was synthesized according to liter-ture method [13]. A reaction mixture formed from 200 g of sodiumydroxide, 8.4 g of tetrabutylammonium hydrogenosulfate, 200 mlf water, and 125 ml of epichlorohydrin is stirred very vigorouslyt room temperature in a 1 L reactor. Freshly distilled pyrrole (40 g,.6 mol) is added dropwise, with cooling in ice in order to keephe temperature of the mixture between 15 and 20 ◦C. The reactions complete after 5 h. The aqueous phase is extracted with ether.he organic phase is washed with aqueous NaCl solution until neu-rality, dried over a molecular sieve, filtered, and distilled underacuum. The product was identified by means of FTIR-ATR, 1H, and3C NMR spectroscopy (not shown).

.3. Preparation of poly(N-glycidylpyrrole-co-pyrrole) (PEP)

The copolymer poly(N-glycidylpyrrole -co-pyrrole) (PEP) filmas electrochemically prepared on an gold electrode from an aque-

us solution containing 0.05 M pyrrole, 0.05 M N-glycidylpyrrolend 1.0 M p-toluene sulphonic acid sodium salt, at a fixed voltage of.8 V vs. Ag/AgCl. The polymer films were prepared with an injectedharge density of 150 mC cm−2. The thickness of the films obtainedas about 1.5 �m as calculated from the injected charge.

.4. Immobilization of GAox poly(N-glycidylpyrrole-co-pyrrole)opolymer film

Galactose oxidase was immobilized by covalent attachmentn poly(N-glycidylpyrrole-co-pyrrole) coated gold electrode. Func-ional epoxy group carrying copolymer film electrode wasmmersed in 100 mM phosphate buffer (pH 7.5) for 2 h, and trans-

erred to the same fresh medium containing GAox (5.0 mg/ml).mmobilization of GAox on the poly(N-glycidylpyrrole-co-pyrrole)lm was carried out by continuously stirring the reaction mediumt 22 ◦C for 24 h. After this period, electrode was removed fromedium and washed with phosphate buffer (100 mM, pH 7.5).

lymer and immobilization of GAox onto copolymer film.

2.5. Amperometric measurements

All amperometric measurements were carried out at room tem-perature in stirred solutions by applying the desired potential andallowing the steady state current to be reached. Once prepared, theGAox electrodes were immersed in 10 ml of 10 mM PBS solution atpH 7.5 and the amperometric response to the addition of a knownamount of galactose solution was recorded.

3. Results and discussion

3.1. Preparation of enzyme electrode

Our approach is the synthesis and biosensor application of thenew epoxy-functionalized polypyrrole derivative. The structureof the new epoxy-functionalized pyrrole monomer is shown inFig. 1. The N-glycidylpyrrole monomer was prepared, at below20 ◦C and under an argon atmosphere, by using tetrabutylam-monium hydrogenosulfate as a phase transfer reagent in waterand purified by vacuum distillation. After preparation of the filmby electropolymerization, GAox was covalently attached onto theelectrode surface.

The constructed enzyme electrode was characterized by FT-IRspectroscopy. Fig. 2 shows FTIR spectra of PEP, PEP-GAox and GAox.The peaks were seen at 1495 cm−1 and 1090 cm−1 in native copoly-mer PEP and PEP-GAox, these have been assigned to C C stretchingmode and C–C stretching, respectively. Also, the epoxide groupgives the band at 905 cm−1. The new peak at 3393 cm−1 in the spec-trum of the PEP-GAox is assigned to N–H deformation which comesfrom GAox’s amide bonds.

The surface morphology of the PEP and PEP-GAox electrodeswere characterized by AFM in order to investigate the homogeneityof the film (Fig. 3). As shown from Fig. 3B, the surface morphology ofthe PEP-GAox electrode is obviously different from that of PEP elec-trode. It could be observed that GAox molecules were covalentlyattached onto the PEP film coated electrode as an aggregated pat-tern in solid-like state with keeping its random cloud-like structureas in bulk solution. From the above results, it could be concludedthat the GAox was immobilized onto the PEP coated electrode.

Fig. 4 shows the typical cyclic voltammograms of the (A) PPyand (B) PEP during electropolymerization in acetonitrile at scanrate 50 mV/s vs. Ag/AgCl. The molar ratio of Py:EpoxyPy was to be1:1. In Fig. 4(A), the starting oxidation peak of pyrrole was appearedat +0.8 V, and the doping/dedoping processes of PPy were observed

442 M. Senel et al. / Synthetic Metals 161 (2011) 440–444

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Fig. 4. Cyclic voltammograms of PPy and PEP during electropolymerization in ace-

Fig. 2. FTIR-ATR spectra of PEP, PEP-GAox and GAox.

y the appearance of redox peaks at +0.7 V and +0.2 V in the CVs ofPy. The peak current of redox peaks increased continuously withncreasing potential cycles and implying the continuous build upf PPy on the working electrode. In order to introduce epoxy groups an reactive site for immobilization of enzyme, PEP was coatedn the surface of gold electrode. In Fig. 4(B), the starting oxidationeak of comonomer with Py and EpoxyPy was appeared at +1.15 V,nd the doping/dedoping processes of PPy were observed by theppearance of redox peaks at +1.1 V and +0.5 V in the CVs of PEP. Theeak current of redox peaks increased continuously with increasingotential cycles and implying the continuous build up of PEP on theorking electrode.

.2. Optimum pH and temperature

The pH effect on the amperometric response of galactose

mmobilized electrode was investigated by measuring the currentesponse to galactose. The effect of the pH on the enzyme electrodeas presented in Fig. 5A. The current increases gradually from pH

.0 to 9.0 and after achieving the maximum current at pH 7.5, the

tonitrile, � = 50 mV/s vs. Ag/AgCl.

Fig. 3. AFM images of (A) PEP and (B) PEP-GAox.

M. Senel et al. / Synthetic Metals 161 (2011) 440–444 443

Fig. 5. (A) Effect of the pH on the current response of enzyme electrode to galactosesmpe

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olution at an applied potential +0.7 V; (B) effect of temperature on the ampero-etric response of enzyme electrode to galactose solution in 10 mM PBS solution,

H 7.5 at an applied potential of +0.7 V vs. Ag/AgCl. Inset was i vs. T−1 plot for thenzyme electrode.

urrent decreases, indicating that the optimum pH 7.5 can be usedor the detection of galactose.

Temperature is the another effect on the activity of the enzy-atic reactions. The activity of the enzyme electrode has been

nvestigated using galactose solution in 10 mM PBS, pH 7.5 solution

y amperometric measurements at temperature varying from 25 to5 ◦C, shown in Fig. 6. It was observed that the response increasedith temperature increase, reaching a maximum at 35 ◦C, and thenecreased. This could have been caused by denaturation of GAoxr film instability at the higher temperatures.

able 1omparison of the analytical performance of the galactose biosensor.

Electrode RT(s) Linear range

PEP 5 2.0–16 mMPGVFc 5 2.0–20 mMPMP 4 2.0–20 mMPVF 30 1.0–40 mMPPy 40 0–24 mM

T, response time; PGVFc, poly (glycidyl methacrylate-co-vinylferrocene); PMP, poly (4-m

Fig. 6. Amperometric response of enzyme electrode to successive addition of 2 mMgalactose solution at an applied potential +0.7 V in stirred 10 mM PBS. The insetshows the calibration curve.

The dependence of amperometric current on temperature in aninitial region can be expressed as an Arrhenius relationship;i(T) =i0 exp

{−EaRT

}where i0 represents a collection of currents, R is the

gas constant, T is the temperature in Kelvin degrees, and Ea is theactivation energy. The activation energy for enzymatic reaction iscalculated to be 4.33 kJ mol−1 from the slope of I − 1/T in the adop-tive region of temperature (inset of Fig. 5B).

3.3. Constant potential response of GAox electrode

Galactose oxidase converts galactose to galactohexodialdoseenzymatically. The enzymatic reaction of galactose may be writtenas follows:

D-galactose + H0O + O2GAOX−→ galactohexodialdose + H2O2

H2O20.7V−→O2 + 2e−

The hydrogen peroxide generated is detected at the workingelectrode at 0.7 V versus Ag/AgCl electrode. Fig. 6 shows the con-stant potential current time response and calibration curve graphsof the prepared enzyme electrode. The linear range can be observedto be up to 16 mM with correlation coefficient (R) of 0.99078, andthen a plateau is reached gradually at the higher galactose con-centration. The biosensor has a good detection limit of 25 �M(signal-to-noise = 3), a high sensitivity of 1.75 �A/mM and a shortresponse time (within ∼5 s).

app

The Michaelis–Menten constant (Km ), determined from Fig. 6using an electrochemical Lineweaver–Burk plot, was 14.7 mM. Thisvalue is much smaller than in earlier studies [handan gülceninyayını ve bizim yayın ve bizim makale], indicating that the presentelectrode exhibits a higher affinity for galactose. In Table 1 the ana-

Detection limit Sensitivity Ref.

25 �M 1.75 �A/mM This work0.1 mM 23 nA/mM [12]– 15 nA/mM [9]– 4.5 �A/mM [14]– 106 nA/mM [15]

ethoxyphenol); PVF, polyvinylferrocene; PPy, polypyrrole.

444 M. Senel et al. / Synthetic Meta

Fig. 7. (A) Reusability of the galactose biosensor. Each data point represents theaverage of data collected by three electrodes (pH 7.5; ∼25 ◦C). (B) Storage stabilitiesof galactose biosensor. The amperometric responses of these enzyme electrodes areregularly checked during 50 days (pH 7.5; ∼25 ◦C).

Table 2Recovery studies of biosensor for determining galactose.

Coriginal (mM) Cadded (mM) Cfound (mM) Recovery (%)a

2.5 1.0 3.46 98.85.0 2.0 6.91 98.7

lpide

[[

49 (2004) 2479.[12] E. Cevik, M. Senel, M.F. Abasiyanik, Curr. Appl. Phys. 10 (2010) 1313.[13] F. Faverolle, A.J. Attias, B. Bloch, P. Audebert, C.P. Andrieux, Chem. Mater. 10

(1998) 740.[14] H. Gulce, I. Ataman, A. Gülce, A. Yıldız, Enzyme Microb. Technol. 30 (2002) 41.[15] W.J. Sung, Y.H. Bac, Sens. Actuators B 114 (2006) 164.

7.5 3.0 10.22 97.3

10.0 5.0 14.54 96.9

a Recovery (%) = Cfound/(Coriginal + Cadded).

ytical performance of this biosensor is compared with that of therior studies. The analytical characteristics of the enzyme electrode

ndicate that the enzyme was effectively attached into the con-ucting polymer film and that the bioactivity of the immobilizednzyme was well preserved in the enzyme electrode.

ls 161 (2011) 440–444

3.4. Stability, reusability and recovery studies

The operational stability galactose biosensor was obtained byrunning measurements in the same day. Between each subsequentmeasurement electrodes stored at 4 ◦C in the buffer solution for10 min. The first 9 measurements revealed the same response,an activity loss of 50% was observed with the subsequent use(Fig. 7A). The response of the galactose biosensor was measuredof its response to galactose for a period of 50 days. As shownin Fig. 7A the amperometric response of the enzyme electroderemained constant for 10 days, and then an activity loss of 55%was observed.

To demonstrate the analytical applicability of the biosen-sor, the recoveries of four galactose samples were determinedby the standard adding method. The results were satisfac-tory. As listed in Table 2, the recovery rate was in the range96.9–98.8%.

4. Conclusions

In the present work, GAox enzyme electrode was prepared bycovalent attachment of GAox onto the electrochemically synthe-sized conducting film to demonstrate the efficiency of the novelcopolymer. The novel enzyme electrode seems to be simple toprepare, fast to respond, inexpensive and sensitive. The analyti-cal characteristics of the enzyme electrode showed that the novelconducting polymer film used in this study is an effective platformto produce reliable biosensors.

Acknowledgment

This research was supported by grants from T.R. Prime MinistryState Planning Organization.

References

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