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Sensors and Actuators B 145 (2010) 444–450

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

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

mperometric hydrogen peroxide biosensor based on covalent immobilization oforseradish peroxidase on ferrocene containing polymeric mediator

ehmet Senela,c,∗, Emre Cevika, M. Fatih Abasıyanıkb,c

Department of Chemistry, 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, TurkeyBiotechnology Research Laboratory, Bionanotechnology Research Center, Fatih University, B.Cekmece, Istanbul 34500, Turkey

r t i c l e i n f o

rticle history:eceived 18 September 2009eceived in revised form6 December 2009ccepted 17 December 2009

a b s t r a c t

In this work, a new type of amperometric hydrogen peroxide biosensor was fabricated for the determina-tion of H2O2. Horseradish peroxidase (HRP) was immobilized on a glassy carbon electrode by poly(glycidylmethacrylate-co-vinylferrocene) (poly(GMA-co-VFc)) film. A polymeric electron transfer mediator, con-taining copolymers of glycidyl methacrylate (GMA) and vinylferrocene (VFc) with different molar ratios,was prepared by free-radical copolymerization. The amperometric response was measured as a func-

vailable online 29 December 2009

eywords:orseradish peroxidaseiosensoredox polymer

tion of H2O2 concentration, at a fixed potential of +0.35 V vs. Ag/AgCl in phosphate-buffered saline (pH7.0). The mediated hydrogen peroxide biosensor showed a fast response of less than 4 s of linear range2.0–30.0 mM, with a detection of 2.6 �M. The sensitivity of the biosensor for H2O2 was 10.42 nA/mM cm2.

© 2009 Elsevier B.V. All rights reserved.

ediator

errocene

. Introduction

Hydrogen peroxide (H2O2) is a byproduct of several highlyelective oxidases, and also an essential mediator in food, biol-gy, medicine, industry and environmental analysis [1,2]. Theevelopment of reliable, sensitive and accurate methods for hydro-en peroxide determination is assuming practical importance.any techniques have been employed for this determination, such

s titrimetry [3], spectroscopy [4], and chemiluminescence [5];owever, these techniques suffer from interferences, long anal-sis time, and involve expensive reagents. Of these techniques,he electrochemical technique based on enzyme biosensors haseen extensively employed for the determination of H2O2 withimplicity, intrinsic selectivity and sensitivity [6–9]. Horseradisheroxidase (HRP) has been most extensively studied in the devel-pment of enzyme-based amperometric biosensors due to its easyvailability in high purity and low cost [10,11]. HRP is an impor-

ant peroxidase that contains a heme group, the protein activeite, with the resting state of the heme ion, Fe(III), as prostheticroup. It can catalyze the H2O2-dependent one-electron oxida-ion of a great variety of substrates [12]. However, to improve the

∗ 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).

925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.snb.2009.12.055

performance and long-term stability of the enzyme electrode, effec-tive immobilization of HRP onto the transducer surface through asuitable matrix is of great significance. Therefore, several valuableimmobilization strategies and materials have been used, includ-ing adsorption [13], crosslinking [14], layer-by-layer assembly [15],sol–gel entrapment [16], surfactant-enzyme complexation [17],biological membranes [18], covalent binding [19], and so on.

Most of the amperometric biosensors described above are basedon the detection of enzymatically produced H2O2. Due to the highworking potential necessary for the direct oxidation of H2O2, cou-pling of oxidases with H2O2-converting enzymes like peroxidaseshas attracted considerable attention [20,21]. This very promisingapplication has motivated a closer investigation of sensor con-struction, with optimized electron transfer pathways between theperoxidases and electrodes. To facilitate the direct electron transferbetween enzyme and electrode, HRP had been immobilized exten-sively onto colloidal Au [22], a biomembrane-like surfactant [23],carbon nanotube [11], a conducting polymer [24,25], polyethyleneglycol [26], nano-Au [28] and others. To date, the immobilizingmatrix is still a significant factor in achieving the direct electro-chemistry of proteins or enzymes.

Ferrocene derivatives are excellent electron transfer mediators,

widely used as mediators to construct mediated amperometricbiosensors. The development of a polymeric mediator for appli-cations in sensor/biosensor is essential because polymers allowthe incorporation of reagents to achieve reagentless devices.Direct attachment of the ferrocene-based mediators onto poly-

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M. Senel et al. / Sensors and

eric films prevents leaching of the mediator. Some examplesf redox copolymers trialing the covalent attachment of fer-ocene are poly(vinylferrocene-co-hydroxyethyl methacrylate)28], poly(N-acryloylpyrrolidine-co-vinylferrocene) [29], acrylmide copolymers [30], and multiwall carbon nanotubes [31].ensitive detection of peroxides is possible using the HRP, fol-owing either direct [32] or mediator-assisted [33,34] electronransfer from HRP. Sensitivity of HRP-based electrodes can be sig-ificantly improved by using mediators [35], like ferrocenes whichave proven their worth in HRP-based electrode [36,37]. HRP and

errocene have attached/immobilized to the electrode surface toonstruct reagentless biosensors [38].

This paper describes the fabrication of the H2O2-sensitiveiosensor, which is based on the one-step immobilization method—he covalent HRP attachment on poly(glycidyl methacrylate-co-inylferrocene). A series of redox copolymers of poly(glycidylethacrylate-co-vinylferrocene) were prepared by the free-radical

opolymerization of vinylferrocene monomer with an epoxy grouparrying a comonomer (GMA), and HRP was immobilized via themine group on the copolymer [39]. Optimized experimental con-itions for the fabrication and operation of the biosensor weretudied. The resulting biosensor has some advantages such as highensitivity, good repeatability and reproducibility.

. Materials and methods

.1. Materials

HRP (EC 1.11.1.7, RZ > 3.0, 250 U/mg) was obtained from Sigma.lycidyl methacrylate (GMA) and 2,2′-azobis (isobutyronitrile)

AIBN), purchased from Fluka and Across Chemical Co., were usedithout further purification. Reagent-grade vinylferrocene (VFc),urchased from Aldrich Co., was used without purification. All otherhemicals were of analytical grade and used without further purifi-ation.

Fig. 1. (a) Preparation of poly(GMA-co-VFc) and (b) immobilization of

tors B 145 (2010) 444–450 445

2.2. Preparation of poly(GMA-co-VFc)

The redox polymer, having different compositions, was pre-pared according to our earlier study (Fig. 1) [39]. A mixtureof VFc and GMA at a known molar ratio was injected into aPyrex tube, AIBN (1 mol% based on the total monomer concen-tration was identical for all samples, 5 mol/dm3). The mixturewas degassed with Argon gas and vacuum sealed. Next, thetubes were placed in constant temperature baths, controlled to70 ◦C. After 2 days, the reaction mixture was added drop wise,while rapidly stirring the diethyl ether to precipitate the copoly-mer. The precipitated copolymer was washed with diethyl etherand reprecipitated in this manner, twice, and finally vacuumdried.

2.3. Preparation of enzyme electrodes

To evaluate the electrochemical property, 10 �L of 1 wt%poly(GMA-co-VFc) in DMF was dropped onto the glassy carbonelectrode. After drying at room temperature, the cyclic voltam-metry (CV) of the copolymer was measured in phosphate-buffered0.8% saline (100 mM PBS, pH 7.0).

HRP was immobilized by covalent attachment on poly(GMA-co-VFc) coated glassy carbon electrode (GCE). 1 wt% solution ofpoly(GMA-co-VFc) in DMF was dropped onto the electrode andair dried. Functional epoxy group carrying copolymer film elec-trode was immersed in phosphate buffer (100 mM, pH 7.0) for2 h, and transferred to the same fresh medium containing HRP(2.0 mg/ml). Immobilization of HRP on the poly(GMA-co-VFc) filmwas carried out by continuously stirring the reaction medium

at 4 ◦C for 24 h. After this period, electrode was removed frommedium and washed with phosphate buffer (100 mM, pH 7.0).Electrochemical properties (CVs) of poly(GMA-co-VFc) and enzymeelectrodes were measured in 10 mM PBS, pH 7.0 at a scan rate5 mV/s.

enzyme via amine group onto poly(GMA-co-VFc) film electrode.

4 Actuators B 145 (2010) 444–450

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46 M. Senel et al. / Sensors and

.4. Electrochemical measurements

Electrochemical measurements were performed using a CHIodel 842B electrochemical analyzer. A small glassy carbon work-

ng electrode (3 mm diameter), a platinum wire counter electrode0.2 mm diameter), Ag/AgCl-saturated KCl reference electrode, andonventional three-electrode electrochemical cell were purchasedrom CH Instruments.

All amperometric measurements were carried out at room tem-erature. They were performed in stirred solutions by applyinghe desired potential and allowing the steady-state current to beeached. Once prepared, the HRP electrode was immersed in 10 mlf a 10 mM PBS, pH 7.0 solution, and the amperometric responseso the addition of known amount of hydrogen peroxide solutionere recorded, respectively. The data are the averages of threeeasurements.

. Results and discussion

.1. Preparation of poly(GMA-co-VFc)

Poly(GMA-co-VFc), possessing different monomer ratios,as prepared from glycidyl methacrylate and vinylferroceneonomers [39]. The present method was effective as the reactive

poxy group was readily introduced into polymer support withoutny modification. Also, the required functional group compositionould also be fixed by changing the comonomer ratio in theolymer preparation mixture.

The epoxy group can bind the protein molecules via their amine,hiol, hydroxyl and carboxyl groups in the pH range where thenzyme is stable and does not lose its activity. The C–N or O–Conds formed between the epoxy groups and biomolecules are sta-le, so that the epoxy group containing supports assists in enzyme

mmobilization. The chemical structure of poly(GMA-co-VFc) andhe immobilization reaction via the amine groups are presented inig. 1.

.2. Cyclic voltammograms of poly(GMA-co-VFc) and HRPmmobilized electrodes

The cyclic voltammograms of poly(MTM-co-VFc) were obtainedetween 0.1 and 0.8 V in a PBS after the copolymer was casted onto

GCE. Fig. 2a shows that the anodic and cathodic currents of the

opolymer film rise continuously with potential scans until a dis-inct redox couple of Fc was attained, and reached steady state after2–16 cycles. After equilibrium was established, the peak potentialpa and Epc values remained constants. The steady-state values for

ig. 2. Cyclic voltammograms of poly(GMA-co-VFc) in 10 mM PBS, pH 7.0 (a) at scan rate 5

Fig. 3. CVs of the enzyme electrode at 10 mM PBS, pH 7.0 in the absence (a) andpresence of 10 mM H2O2 (b). Scan rate: 50 mV/s.

the Epa and Epc of cyclic voltammograms shown in Fig. 2a, are 0.54and 0.33 V, respectively.

Typical cyclic voltammograms of the electrode loaded witha copolymer in PBS, when the scan rate was altered from 1 to20 mV/s, are shown in Fig. 2b. A linear correlation between theanodic peak current, Ipa, and square root of scan rate, v1/2, wasobtained, indicating that charge propagation in the polymer occursby a diffusion-like process, such as electron hopping among neigh-boring redox sites and counter-ion motion. Initially, the ferric ion inthe VFc units exists in both the reduced form (Fe(II)) and oxidizedform (Fe(III)). During the forward scan, Fe(II) is oxidized to Fe(III),and subsequently an oxidation current peak is observed. During thereverse scan, Fe(III) is reduced. The difference of the redox peakswas increased with an increase in the scan rate. The voltammetricbehavior also indicates that ferrocene has been immobilized on thesurface of the glassy carbon electrode.

3.3. Electrochemical response to hydrogen peroxide at thebiosensor

The electrocatalytical reactivity of the enzyme electrodetowards H2O2 was investigated by cyclic voltammetry. Fig. 3 showsthe current response of a poly(GMA-co-VFc)/HRP enzyme electrodein the absence and presence of H2O2. The observed increase in

mV/s (b) at scan rate of 1, 2.5, 5, 7.5, 10, 15 and 20 mV/s (from internal to external).

M. Senel et al. / Sensors and Actuators B 145 (2010) 444–450 447

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performance of this biosensor is compared with that of the prior

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ig. 4. Comparison of amperometric responses of the enzyme electrode fabricatedith (a) GMA-co-VFc0.2, (b) GMA-co-VFc0.4, (c) GMA-co-VFc0.6, and (d) GMA-co-Fc0.8 to successive addition of H2O2 at constant potential 10 mM PBS, pH 7.0. Insetlot of amperometric response of different electrodes vs. H2O2 concentration.

nodic current, in the presence of H2O2, indicates that the catalyticeaction enhances the oxidation current of the enzyme electroden a manner similar to that reported earlier [16]. This result implieshat the enzyme-dependent catalytic current response of H2O2hich originated from the HRP reaction was mediated by the

opolymer Fc units. Thus, the copolymer could effectively shuttlelectrons from the redox center of HRP to the electrode surface.he reaction mechanism of the biosensor can be summarized asollows:

First, HRP reduces hydrogen peroxide to water.

2O2 + HRP → H2O + HRP(I)

Then, HRP can be regenerated by using a mediator through twoeparate one-electron steps,

RP(I) + Fc(red) → HRP(II) + Fc(ox)

RP(II) + Fc(red) → HRP + Fc(ox)

c(ox) + 2e− → Fc(red)

Finally, the Fc can be recycled at the electrode as the mediator,roducing an increase in its reduction current.

.4. Catalytic current for HRP immobilized electrodes

The steady-state catalytic current responses of the enzyme elec-rodes containing GMA and VFc of different compositions as aunction of hydrogen peroxide concentration at +0.35 V vs. Ag/AgClre shown in Fig. 4. The catalytic current of the enzyme electrode

able 1omparison of the analytical performance of the H2O2 biosensor based on HRP.

Electrode RT(s) Linear range

GCE/P(GMA-co-VFc) 4 2.0–30 mMGCE/P(m-AAMFc) – 0.08–15 �MGCE/MDMS – 0.2–680 �MGCE/CS-THEOS 5 1.0–250 �MGCE/FBCS 20 35–2000 �M

CE, glassy carbon electrode; RT, response time; P(m-AAMFc), poly(m-aminoanilinometerrocene branched chitosan.

Fig. 5. Amperometric responses of enzyme electrode at an applied potential +0.35 Vto successive glucose injections in a stirred 10 mM PBS, pH 7.0. The Lineweaver–Burkplot and calibration curve is shown inset.

increases with an increase in the hydrogen peroxide concentra-tions. Apparently, this behavior follows the Michaelis–Mentenkinetics, as seen in several amperometric enzyme electrodes [40].The inset of Fig. 4 shows that the catalytic current responsedepends on the copolymer constituents. The catalytic response ofthe enzyme electrodes reaches a maximum by increasing the VFccomposition up to 0.4 ratios.

3.5. Amperometric determination of hydrogen peroxides

The amperometric measurement of H2O2 at the GC/poly(GMA-co-VFc)/HRP electrode has been investigated, and the calibrationcurve of the response current of the enzyme electrode to H2O2concentration is shown in Fig. 5. The inset plot shows the responsecurrent of the successive addition of 2.0 mM H2O2. From Fig. 5, thelinear range can be observed to be up to 30 mM with correlationcoefficient (R) of 0.9984, and then a plateau is reached gradu-ally at the higher H2O2 concentration. The biosensor has a gooddetection limit of 2.6 �M (signal-to-noise = 3), a high sensitivity of10.42 nA/mM cm2 and a short response time (within ∼4 s).

The Kappm was determined by analyzing the slope and intercept

for the plot of the reciprocals of the cathodic current vs. H2O2 con-centration. The apparent Michaelis–Menten constant Kapp

m in thepresent study is calculated to be 1.14 mM. In Table 1 the analytical

studies. This value was much smaller than in earlier studies [41],indicating that the present electrode exhibits a higher affinity forH2O2. The covalent immobilization of HRP on the copolymer filmfor the fabrication of as-prepared electrode appears to enhance andimprove the biosensor performance.

Detection limit Sensitivity Ref.

2.6 �M 10.42 nA/mM cm2 This work0.08 �M 34 nA/�M [42]0.078 �M – [12]0.4 �M – [43]15 �M – [44]

hylferrocene); CS, chitosan; THEOS, tetrakis(2-hydroxyethyl) orthosilicates; FBCS,

448 M. Senel et al. / Sensors and Actuators B 145 (2010) 444–450

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ig. 6. Influences of pH values of PBS solution on the peak current of the responseurrent to 10 mM H2O2 at the enzyme electrode.

.6. The effect of the pH

The effect of the pH value of the solution on the enzyme elec-rode response has been investigated to be between 5.0 and 8.0,ith the corresponding results shown in Fig. 6. From Fig. 6, the

esponse current of the enzyme electrode is observed to increaseith an increase in the pH value, and the maximum response cur-

ent is seen at pH 7.0, in agreement with that the results reported inhe literature [45]. Therefore, pH 7.0 is used in further experimentsnd determination of H2O2.

.7. The effect of the temperature and determination of activationnergy

The effect of temperature on the steady-state amperometricesponse was also investigated in the range of 30–50 ◦C, shownn Fig. 7. The response increased with temperature increase, reach-ng a maximum at 45 ◦C, and then decreased. This could have beenaused by denaturation of HRP or film instability at the higher tem-eratures.

The dependence of amperometric current on temperature in annitial region can be expressed as an Arrhenius relationship

(T) = i0 exp{−Ea

RT

}

ig. 8. (a) Operational stability and (b) storage stabilities of HRP immobilized electrodeuring 30 days (pH 7.0; ∼25 ◦C).

Fig. 7. The effect of the temperature on the response of the enzyme electrode to10 mM H2O2 in 10 mM PBS, pH 7.0 solution. Inset was i vs. T−1 plot for the enzymeelectrode.

where i0 represents a collection of currents, R is the gas constant,T is the temperature in Kelvin degrees, and Ea is the activationenergy. The activation energy for enzymatic reaction is calculatedto be 1.67 kJ mol−1 from the slope of I–1/T in the adoptive regionof temperature (inset of Fig. 7). This Ea value obtained is smallerthan those reported by Xu et al. [46] for HRP immobilized on thepolyaniline films. The smaller Ea value means that the HRP immo-bilized on the poly(GMA-co-VFc) film possesses higher enzymaticactivity.

3.8. Operational, storage stabilities and recovery studies

The operational stability HRP electrode obtained by run-ning measurements on the same day. Between each subsequentmeasurement, the electrodes were stored at 4 ◦C in the buffersolution for 20 min. The first 15 measurements revealed the sameresponse, an activity loss of 30% was observed with subsequent use(Fig. 8a).

The response of the HRP electrode was measured by its reac-tion to 10 mM H2O2 for a period of 60 days. As shown in Fig. 8b,the amperometric response of the enzyme electrode remainedalmost constant for 30 days, followed by 50% activity loss. Covalentattachment of the HRP on the electrode could be responsi-

. The amperometric responses of these enzyme electrodes are regularly checked

M. Senel et al. / Sensors and Actua

Table 2Recovery studies of biosensor for determining H2O2.

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

0.50 0.25 0.758 101.12.50 1.0 3.486 99.65.0 2.5 7.324 97.7

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le for protecting the enzyme from environmental effects andeaking.

To demonstrate the analytical applicability of the biosensor, theecoveries of four H2O2 samples were determined by the standarddding method. The results were satisfactory. As listed in Table 2,he recovery rate was in the range 94.4–101.1%.

. Conclusions

Poly(GMA-co-VFc), as a polymeric redox mediator, has beenevealed as an attractive material for immobilization of HRP to con-truct the hydrogen peroxide biosensor. Immobilized HRP on theoly(GMA-co-VFc) films maintains its activity. A reliable, low-costnd sensitive biosensor for hydrogen peroxide detection is thuseveloped, possessing a variety of excellent characteristics, includ-

ng high sensitivity, good repeatability and reproducibility, rapidesponse and long-term stability.

cknowledgements

This research was supported by grants from T.R. Prime Ministrytate Planning Organization and Fatih University Research Supportffice (P50090801-1).

eferences

[1] T. Ruzgas, E. Csöregi, J. Emneus, L. Gorton, G. Marko-Varga, Peroxidase-modified electrodes: fundamentals and application, Anal. Chim. Acta 330(1996) 123–138.

[2] L. Wang, E. Wang, A novel hydrogen peroxide sensor based on horseradishperoxidase immobilized on colloidal Au modified ITO electrode, Electrochem.Commun. 6 (2004) 225–229.

[3] E.C. Hurdis, J.H. Romeyn, Accuracy of determination of hydrogen peroxide bycerateoxidimetry, Anal. Chem. 26 (1954) 320–325.

[4] C. Matsubara, N. Kawamoto, K. Takamura, Oxo[5,10,15, 20-tetra(4-pyridyl)-porphyrinato]titanium (IV): an ultra-high sensitivity spectrophotometricreagent for hydrogen peroxide, Analyst 117 (1992) 1781–1784.

[5] K. Nakashima, K. Maki, S. Kawaguchi, S. Akiyama, Y. Tsukamoto, K. Imai, Per-oxyoxalate chemiluminescence assay of hydrogen peroxide and glucose using2,4,6,8-tetrathiomorpholinopyrimido[5,4-d]pyrimidine as a fluorescent com-ponent, Anal. Sci. 7 (1991) 709–713.

[6] J. Zhang, M. Oyama, Gold nanoparticle-attached ITO as a biocompatible matrixfor myoglobin immobilization: direct electrochemistry and catalysis to hydro-gen peroxide, J. Electroanal. Chem. 577 (2005) 273–279.

[7] E. Ferapontova, K. Schmengler, T. Börchers, T. Ruzgas, L. Gorton, Effect of cys-teine mutations on direct electron transfer of horseradish peroxidase on gold,Biosens. Bioelectron. 17 (2002) 953–963.

[8] J. Wang, L. Wang, J. Di, Y. Tu, Electrodeposition of gold nanoparticles onindium/tin oxide electrode for fabrication of a disposable hydrogen peroxidebiosensor, Talanta 77 (2009) 1454–1459.

[9] J. Hong, Z. Dai, Amperometric biosensor for hydrogen peroxide and nitrite basedon hemoglobin immobilized on one-dimensional gold nanoparticle, Sens. Actu-ators B: Chem. 140 (2009) 222–226.

10] L. Qjan, X. Yang, Composite film of carbon nanotubes and chitosan for prepa-ration of amperometric hydrogen peroxide biosensor, Talanta 68 (2006)721–727.

11] J.L. Huang, Y.C. Tsai, Direct electrochemistry and biosensing of hydro-gen peroxide of horseradish peroxidase immobilized at multiwalled carbon

nanotube/alumina-coated silica nanocomposite modified glassy carbon elec-trode, Sens. Actuators B: Chem. 140 (2009) 267–272.

12] H.L. Zhang, G.S. Lai, D.Y. Han, A.M. Yu, An amperometric hydrogen peroxidebiosensor based on immobilization of horseradish peroxidase on an electrodemodified with magnetic dextran microspheres, Anal. Bioanal. Chem. 390 (2008)971–977.

[

tors B 145 (2010) 444–450 449

13] Y. Xiao, H.X. Ju, H.Y. Chen, Direct electrochemistry of horseradish peroxidaseimmobilized on a colloid/cysteamine-modified gold electrode, Anal. Biochem.278 (2000) 22–28.

14] L. Qian, X. Yang, Direct electrochemistry of horseradish peroxidase immo-bilized on a colloid/cysteamine-modified gold electrode, Talanta 68 (2006)721–727.

15] P. He, N. Hu, Electrocatalytic properties of heme proteins in layer-by-layerfilms assembled with SiO2 nanoparticles, Electroanalysis 16 (2004) 1122–1131.

16] G. Wang, J.J. Xu, H.Y. Chen, Z.H. Lu, Amperometric hydrogen peroxide biosen-sor with sol–gel/chitosan network-like film as immobilization matrix, Biosens.Bioelectron. 18 (2003) 335–343.

17] S. Mahiuddin, A. Renoncourt, P. Bauduin, D. Touraud, W. Kunz, Horseradishperoxidase activity in a reverse catanionic microemulsion, Langmuir 21 (2005)5259–5262.

18] P.B. Beata, M. Jan, L. Andrzej, Conducting polymer films as model biologi-cal membranes: electrochemical and ion-exchange properties of poly(pyrrole)films doped with asparagine and glutamine, Electrochim. Acta 51 (2006)2173–2181.

19] S. Salomão, C. Kátia, F. Fernandes, Covalent immobilization of horseradish per-oxidase onto poly(ethylene terephthalate)–poly(aniline) composite, ProcessBiochem. 39 (2004) 883–888.

20] A. Lindgren, J. Emnéus, T. Ruzgas, L. Gorton, G. Marko-Varga, Amperomet-ric detection of phenols using peroxidase-modified graphite electrodes, Anal.Chim. Acta 357 (1997) 51–62.

21] T. Ruzgas, L. Gorton, J. Emnéus, G. Marko-Varga, Kinetic models of horseradishperoxidase action on a graphite electrode, J. Electroanal. Chem. 391 (1995)41–49.

22] J. Jia, B. Wang, A. Wu, G. Cheng, Z. Li, S.J. Dong, A method to constructa third-generation horseradish peroxidase biosensor: self-assembling goldnanoparticles to three-dimensional sol–gel network, Anal. Chem. 74 (9) (2002)2217–2223.

23] X.H. Chen, X.S. Peng, J.L. Kong, J.Q. Deng, Facilitated electron transfer from anelectrode to horseradish peroxidase in a biomembrane-like surfactant film, J.Electroanal. Chem. 480 (2000) 26–33.

24] Y.T. Kong, M. Boopathi, Y.B. Shim, Direct electrochemistry of horseradish per-oxidase bonded on a conducting polymer modified glassy carbon electrode,Biosens. Bioelectron. 19 (2003) 227–232.

25] X.L. Luo, A.J. Killard, A. Morrin, M.R. Smyth, Enhancement of a conduct-ing polymer-based biosensor using carbon nanotube-doped polyaniline, Anal.Chim. Acta 575 (2006) 39–44.

26] Y. Xu, W.L. Peng, X.J. Liu, G.X. Li, A new film for the fabrication of an unmediatedH2O2 biosensor, Biosens. Bioelectron. 20 (2004) 533–537.

28] T. Saito, M. Watanabe, Characterization of poly(vinylferrocene-co-2-hydroxyethyl methacrylate) for use as electron mediator in enzymatic glucosesensor, React. Funct. Polym. 37 (1998) 263–269.

29] S. Koide, K. Yokoyama, Electrochemical characterization of an enzyme elec-trode based on a ferrocene-containing redox polymer, J. Electroanal. Chem.468 (1999) 193–201.

30] N. Kuramoto, Y. Shishido, K. Nagai, Preparation of thermally responsive andelectroactive poly(N-acryloylpyrrolidine-co-vinylferrocene), Macromol. RapidCommun. 15 (1994) 441–444.

31] V.B. Kandimalla, V.S. Tripathi, H. Ju, A conductive ormosil encapsulated with fer-rocene conjugate and multiwall carbon nanotubes for biosensing application,Biomaterials 27 (2006) 1167–1174.

32] E. Csöregi, L. Gorton, G. Marko-Varga, Carbon fibres as electrode materials forthe construction of peroxidase-modified amperometric biosensors, Anal. Chim.Acta 273 (1993) 59–70.

33] T.J. Ohara, M.S. Vreeke, F. Battaglini, A. Heller, Bienzyme sensors based on elec-trically wired peroxidase, Electroanalysis 5 (1993) 825–831.

34] M. Ashok, P. Shengtian, Ferrocene-conjugated m-phenylenediamine conduct-ing polymer-incorporated peroxidase biosensors, Anal. Biochem. 267 (1999)141–147.

35] T. Tatsuma, M. Gondaira, T. Watanabe, Peroxidase-incorporated polypyrrolemembrane electrode, Anal. Chem. 64 (1992) 1183–1187.

36] P.D. Sanchez, A.J.M. Ordieres, A.C. Garcia, P.T. Blanco, Peroxidase-ferrocenemodified carbon paste electrode as an amperometric sensor for the hydrogenperoxide assay, Electroanalysis 3 (1991) 281–285.

37] A.L. Crumbliss, J.G. Stonehuerner, R.W. Henkens, J. Zhao, J.P. O’Daly, Acarrageenan hydrogel stabilized colloidal gold multi-enzyme biosensorelectrode utilizing immobilized horseradish peroxidase and cholesterol oxi-dase/cholesterol esterase to detect cholesterol in serum and whole blood,Biosens. Bioelectron. 8 (1993) 331–337.

38] P.C. Pandey, S. Upadhyay, B. Upadhyay, Peroxide biosensors and mediatedelectrochemical regeneration of redox enzymes, Anal. Biochem. 252 (1997)136–142.

39] F. Dursun, S. K. Ozoner, A. Demirci, M. Gorur, B. Keskinler, F. Yilmaz, E. Erhan,The use of vinylferrocene based copolymers for the fabrication of tyrosinaseworking electrodes, Biomacromolecules, submitted.

40] S. Chen, R. Yuan, Y. Chai, B. Yin, W. Li, L. Min, Amperometric hydrogen peroxide

biosensor based on the immobilization of horseradish peroxidase on core–shellorganosilica@chitosan nanospheres and multiwall carbon nanotubes compos-ite, Electrochim. Acta 54 (2009) 3039–3046.

41] Z. Tong, R. Yuan, Y. Chai, Y. Xie, S. Chen, A novel and simple biomolecules immo-bilization method: electro-deposition ZrO2 doped with HRP for fabrication ofhydrogen peroxide biosensor, J. Biotechnol. 128 (2007) 567–575.

4 Actua

[

[

[

[

[

50 M. Senel et al. / Sensors and

42] A. Mulchandani, S. Pan, Ferrocene-conjugated m-phenylenediamine conduct-ing polymer-incorporated peroxidase biosensors, Anal. Biochem. 267 (1999)141–147.

43] G.H. Wang, L.M. Zhang, Using novel polysaccharide-silica hybrid material toconstruct amperometric biosensor for hydrogen peroxide, J. Phys. Chem. B 110(2006) 24684–24868.

44] A. Garcia, C. Peniche-Covas, B. Chico, B.K. Simpson, R. Villalonga, Ferrocenebranched chitosan for the construction of a reagentless amperometric hydro-gen peroxide biosensor, Macromol. Biosci. 7 (2007) 435–439.

45] G.S. Lai, H.L. Zhang, D.Y. Han, A novel hydrogen peroxide biosensor based onhemoglobin immobilized on magnetic chitosan microspheres modified elec-trode, Sens. Actuators B: Chem. 129 (2008) 497–503.

46] Q. Xu, C. Mao, N.N. Liu, J.J. Zhu, J. Shen, Immobilization of horseradish peroxi-dase on o-carboxymethylated chitosan/sol–gel matrix, React. Funct. Polym. 66(2006) 863–870.

tors B 145 (2010) 444–450

Biographies

Mehmet Senel received his BS and MS degrees in Chemistry from Fatih Universityin 2004 and 2007. He is now a PhD candidate and working as a research assistant atFatih University. He is performing research in the biotechnology research laboratoryon development of biosensors via immobilizing enzymes on polymeric mediators.

Emre Cevik is now an MSc candidate in Institute of Science and Engineering,

Department of Chemistry, Fatih University. His current researches are enzymeimmobilization and electrochemical biosensors.

M. Fatih Abasıyanık is an assistant professor in Department of Genetics and Bioengi-neering, Faculty of Engineering, Fatih University. His current researches are enzymeimmobilization, biosensor, genetics and microbiology.