Cp

MD

a

ARRAA

KPFPGB

1

aottTttuhpsm[io[sotn

0d

Synthetic Metals 161 (2011) 1861– 1868

Contents lists available at ScienceDirect

Synthetic Metals

journa l h o me page: www.elsev ier .com/ locate /synmet

onstruction of reagentless glucose biosensor based on ferrocene conjugatedolypyrrole

ehmet S enelepartment of Chemistry, Faculty of Arts and Sciences, Fatih University, B. Cekmece, Istanbul 34500, Turkey

r t i c l e i n f o

rticle history:eceived 15 April 2011eceived in revised form 14 June 2011ccepted 19 June 2011vailable online 16 July 2011

eywords:olymer

a b s t r a c t

Glucose oxidase (GOx) was covalently immobilized onto an electrochemically prepared novel copoly-mer of ferrocene branched polypyrrole for the construction of an amperometric glucose biosensor.Ferrocene-pyrrole monomers were prepared by condensation reaction between ferrocene ethanol and3-(1H-pyrrol-1-yl)propanoic acid and ferrocene-pyrrole, 3-(1H-pyrrol-1-yl)propanoic acid, and pyrrolemonomers were electrochemically polymerized on the surface of the electrode. GOx was covalentlyimmobilized onto the co-polymer coated electrode surface by using coupling agents to create aminebonds. The prepared copolymers were utilized as conducting films for amperometric glucose sensing

erroceneyrrolelucose oxidaseiosensor

after immobilization of GOx. Amperometric response was measured as a function of concentration ofglucose at a fixed potential of +0.38 V vs. Ag/AgCl in a phosphate-buffered saline solution (pH 7.0). Resultsclearly showed that the ferrocene groups on the co-polymer play the role of electron transfer mediatorbetween the redox center of glucose oxidase (GOx) and the surface of the electrode. Also, the effects ofpH and temperature, storage, reusability and interference of the amperometric glucose biosensor wereinvestigated.

. Introduction

The establishment of electron transfer between a redox enzymend an electrode is an essential prerequisite for the developmentf biosensors [1] and biofuel cells [2]. To establish an electronransfer, researchers used mediators to access the redox cen-er of an enzyme and then to act as the charge carriers [3,4].he electro-active compound was chosen as mediator to improvehe linear range selectivity, sensitivity, and response time ofhe bioelectrodes [5]. In view of the development of a molec-lar device, immobilization of enzymes onto electrode surfacesas been extensively studied. Conducting polymer interfaces arearticularly suitable for localizing biomolecules onto micro-sizedurfaces [6]. Besides, conducting polymers offer the facility toodulate their electronic properties via molecular interactions

7]. Many researchers have suggested that covalent functional-zation of conducting polymers could be achieved by synthesisf functionalized monomers, which are subsequently polymerized8,9].Ferrocene derivatives are widely used as mediators in the con-truction of mediated amperometric biosensors. Direct attachment

f the ferrocene-based mediators onto polymeric films preventshe mediator from leaching. Many investigators have reportedovel redox copolymers where covalent attachment of ferrocene

E-mail address: msenel@fatih.edu.tr

379-6779/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.synthmet.2011.06.025

© 2011 Elsevier B.V. All rights reserved.

has been attempted such as poly(vinylferrocene-co-hydroxyethylmethacrylate) [10], poly(N-acryloylpyrrolidine-co-vinylferrocene)[11], acryl amide copolymers [12], ferrocene-based polyamides[13], and poly(glycidyl methacrylate-co-vinylferrocene) [14],ferrocene-polythiophene derivative [15]. The determination ofglucose is important in the food and fermentation industriesand in clinical chemistry, and there have been many reports onthis subject. The most frequently employed glucose biosensorsare based on glucose oxidase (GOx). In earlier studies, the cat-alytic conversion of glucose to gluconolactone by GOx in thepresence of dioxygen produced hydrogen peroxide which wasthen detected electrochemically. However, there were sensitivityproblems in O2 concentration and significant interference signalsgenerated by ascorbate, urate, and paracetamol [16]. Later, thisproblem was overcome by introducing mediators as means ofelectron transfer instead of using oxygen [6–8]. Recently, due tofundamental interest in electron-transfer reaction between GOxand electrodes and from the perspective of long-term stability ofglucose sensors, several redox-active polymers have been preparedand utilized as polymeric mediators [14–22]. Since it has beenestablished that glucose oxidase could be successfully immobilizedin polypyrrole derivatives [23–25] and that an electron acceptor to

the enzyme, ferrocene, could be easily modified by chemical sub-stitution [26,27], it was hoped that pyrrole-ferrocene conjugatescould be synthesized and used in the construction of reagentlessmediated biosensors.

1 etals 161 (2011) 1861– 1868

1afivcotcm

2

2

m(a(4tFscp

2

fgcrc

2

cowtwepws

2

mgctetF

2

((

Table 1Amounts of GOx immobilized on various copolymer film, apparentMichaelis–Menten constants and maximum current densities for enzymeelectrodes.

Abbreviation(Pyx/PyCO2Hy/PyFcz)a

Amount of ImmobilizedGOx (�g/cm2)

Kmapp (mM) Imax (nA)

Py0.6/PyCO2H0.2/PyFc0.2 114 10.15 177Py0.4/PyCO2H0.2/PyFc0.4 126 4.89 308Py0.4/PyCO2H0.4/PyFc0.2 132 9.62 223Py0.2/PyCO2H0.4/PyFc0.4 119 6.78 325Py0.2/PyCO2H0.2/PyFc0.6 121 4.73 373

862 M. S enel / Synthetic M

In the present paper, a novel copolymer of N-(3-(1H-pyrrol--yl)ethyl)ferrocenecarboxate (Py-Fc) monomer was synthesizednd employed as a novel electron-mediating support material forabricating GOx-immobilized electrodes. For this purpose, the GOx-mmobilized electrodes were prepared with the copolymers ofarious compositions and applied to amperometric sensing of glu-ose. Their sensing ability was investigated in relation to the effectf the copolymer composition on the conductivity of the film andhe immobilization of GOx. It was demonstrated that these redoxopolymers function as an enzyme-binding support and a poly-eric electron-transfer mediator for enzymatic glucose biosensor.

. Experimental

.1. Materials

Glucose oxidase (GOx) (EC 1.1.3.4), 1-cyclohexyl-3-(2-orpholinoethyl)-carbodiimide metho-p-toluenesulfonate

CMC), 1-(2-cyanoethyl)pyrrole (Py-CN), and ferroceneldehyde were obtained from Aldrich Chemical Co. 3-Dimethylamino)propyl)ethyl carbodiimide hydrochloride (EDCI),-dimethylaminopyridine (DMAP), pyrrole monomer (Py), p-oluene sulfonic acid sodium salt, and glucose were procured fromluka. Pyrrole monomer was distilled thrice and the p-tolueneulfonic acid solution was freshly prepared before use. All otherhemicals were of analytical grade and used without furtherurification.

.2. Apparatus

Electrochemical polymerizations and measurements were per-ormed using a CHI Model 842B electrochemical analyzer. A smalllassy carbon working electrode (2 mm diameter), a platinum wireounter electrode (0.2 mm diameter), an Ag/AgCl-saturated KCleference electrode, and a conventional three-electrode electro-hemical cell were purchased from CH Instruments.

.3. Synthesis of 1-(2-carboxyethyl)pyrrole (Py-CO2H)

1-(2-Carboxyethyl)Pyrrole was obtained by hydrolysis of 1-(2-yanoethyl)pyrrole (Py-CN) according to literature [28]: a mixturef 25 g of Py-CN and 100 ml of 15% potassium hydroxide solutionas stirred at 50 ◦C for 40 h. Then the mixture was cooled to room

emperature and acidified by hydrochloric acid. After extractionith ether, the crude product (colorless crystals) was collected on

vaporation of ether. The crude product was dissolved in ether andurified by recrystallization from the ether solution. The productas identified as Py-CO2H by means of FTIR-ATR, 1H, and 13C NMR

pectroscopy (not shown).

.4. Synthesis of ferrocene alcohol

To a solution of ferrocene aldehyde in tetrahydrofuran (THF) andethanol (1:2, v/v), a suspension of sodium borohydride in THF was

radually added by stirring simultaneously. Once the addition wasomplete, the reaction was allowed to take place for 1 h at roomemperature. After the solvent was removed, the crude product wasxtracted by ether and recrystallized from n-hexane. The solid washen dried at 50 ◦C under reduced pressure and characterized usingTIR-ATR, 1H, and 13C NMR spectroscopy (not shown).

.5. Synthesis of Py-Fc monomer

A solution of EDCI (600 mg, 3.14 mmol) in dichloromethane4 ml) was added dropwise to a solution of ferrocene alcohol750 mg, 3.5 mmol), 1-(2-carboxyethyl)pyrrole (500 mg, 3.6 mmol)

Py0.2/PyCO2H0.6/PyFc0.2 146 7.25 260

a x, y and z values are mole fractions of monomers in the feed.

and DMAP (30 mg, 0.3 mmol) in dichloromethane (6 ml) at −10 ◦C.After the addition, the reaction mixture was allowed to stir for 20 hat room temperature under argon atmosphere. The reaction mix-ture was washed thrice with a 1 M sodium hydroxide solution andtwice with distilled water. The organic layer was dried using anhy-drous magnesium sulfate and the solvent was removed in vacuo. Ayellow solid was obtained after crystallization in ethanol.

2.6. Electrochemical polymerization

Electrochemical polymerization was carried out in a solutionof monomers of different concentrations (Py, Py-CO2H, and Py-Fc, Table 1.) and 1.0 M p-toluene sulfonic acid sodium salt at afixed voltage of 1.2 V vs. Ag/AgCl. The copolymer films were pre-pared at an injected charge density of 100 mC/cm2 (except for thespecial requirements). The copolymer Py/Py-CO2H/Py-Fc was thenobtained on the working electrode deposited in the form of a thinfilm. The composition of the copolymer was determined by meansof FTIR-ATR spectroscopy.

2.7. Immobilization of GOx on Py/Py-CO2H/Py-Fc copolymer film

The copolymer film-coated electrode was immersed in 3.0 mldistilled water containing 25 mg GOx and 120 mg CMC, and allowedto remain for 24 h at 4 ◦C. The copolymer films treated thus, i.e.GOx-electrodes, were then rinsed with distilled water and storedin 0.1 M phosphate buffer (pH 7.4) at 4 ◦C.

The amounts of bound protein deposited on the electrodes weredetermined by the Lowry method [29]. The quantity of covalentlybound protein was calculated by subtracting the amounts recov-ered in the combined washings of the enzyme-electrode from theprotein used for immobilization.

2.8. 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, theGOx electrodes were immersed in 10 ml of 10 mM PBS solution atpH 7.4 and the amperometric response to the addition of a knownamount of glucose solution was recorded. The data shown are theaverage of three measurements for each electrode.

3. Results and discussion

3.1. Preparation of Py-Fc monomer

Our approach is for the synthesis and biosensor application

of the new ferrocene-functionalized polypyrrole derivative. Thestructure of the new ferrocene-functionalized pyrrole monomer isshown in Fig. 1. The Py-Fc monomer was prepared, at room tem-perature and under an argon atmosphere, by condensation reaction

M. S enel / Synthetic Metals 161 (2011) 1861– 1868 1863

tion o

oy

1

tsm

Fig. 1. (A) Synthesis of Py-Fc monomer and its copolymer. (B) Immobiliza

f equimolar amounts of ferrocene ethanol and 3-(1H-pyrrol-1-l)propanoic acid in chloroform in the presence of EDCI and DMAP.

The Py-Fc monomer structures have been established by 1H and3 1

C NMR spectroscopy. The H NMR spectra of the monomer exhibithe characteristic peaks of the structure, as shown in Fig. 2. In thepectrum of Py-Fc, key signals arising from the cyclopentadienyloieties are observed in the range 4.18–4.25 ppm; two apparent

Fig. 2. 1H and 13C NMR spec

f enzyme via carboxylic acid group onto copolymer coated film electrode.

triplets corresponding to the � and � protons of the pyrrole arecentered at about 6.16 and 6.66 ppm the resonances due to the ethylchain linked to the pyrrole group are shown at 2.77 and 4.28 ppm.

13

The C NMR spectrum of Py-Fc is also shown in Fig. 2. The res-onance signal at 171 ppm is due to the presence of ester carbonylcarbons. The ethylene carbon atoms of pyrrole and methylene car-bon atom of ferrocene generate signals at 37 (C3), 45 (C4), and 64

tra of Py-Fc monomer.

1864 M. S enel / Synthetic Metals 1

Fig. 3. (a) Cyclic voltammogram of Py-Fc (1 mM) in 0.10 M tetraethylammoniumpga

(pvt

amiFac−ttt

3

1

ota

erchlorate (TEAPC)/CH3CN, � = 100 mV/s vs. Ag/AgCl. (b) Typical cyclic voltammo-rams of Py-Fc monomer; the inset shows the calibration plot of the peak currentt scan rates varied between 5 and 300 mV/s (1 mM) in 0.10 M TEAPC/CH3CN.

C6) ppm, respectively. The carbon atoms of the pyrrole ring dis-lay signals at 108 (C1) and 121 (C2) ppm. The resonance signalsisible at 81 (C7), 69.7 (C8), 69 (C9), and 68.6 (C10) ppm belong tohe cyclopentadienyl group.

The cyclic voltammogram (CV) of Py-Fc in acetonitrile shows reversible redox process at E1/2 0.75 V because the ferroceneoiety is followed by an irreversible wave corresponding to the

rreversible oxidation peak of the pyrrole group at 1.1 V (Fig. 3a).ig. 3b shows the typical cyclic voltammograms of the Py-Fc incetonitrile at different scan rates varying from 5 to 300 mV/s. Well-haracterized redox peaks were observed at a potential range of0.2 to 0.7 V. As shown in Fig. 3b, the peak currents were propor-

ional to the square roots of the scan rates (V1/2), which showed aypical diffusion-controlled electrochemical behavior according tohe literature [30].

.2. Preparation of Py/Py-CO2H/Py-Fc film and enzyme electrode

The structure of Py/Py-CO2H/Py-Fc copolymer was confirmed by

H and 13C NMR spectroscopy (Fig. 4). In the proton NMR spectrumf the copolymer, the peaks between 4.40 and 4.55 ppm are due tohe presence of cyclopentadienyl protons. The peaks between 6.4nd 7.0 ppm are specific resonance signals of pyrrole ring protons.

61 (2011) 1861– 1868

The peak at 8.2 ppm belongs to carboxylic acid proton. The 13C NMRspectrum of Py/Py-CO2H/Py-Fc is shown in Fig. 4. The resonancesignals at 177 and 171.5 ppm are due to the presence of carboxylicacid and ester carbonyl carbons, respectively. The ethylene carbonatoms of pyrrole and the methylene carbon atom of ferrocene gen-erate signals at 44.5 (C5), 36.5 (C6), and 64 (C9) ppm, respectively.The carbon atoms of the substituted and un-substituted pyrrolering display signals between 108 and 121 ppm. The resonance sig-nals visible at 81 (C10), 69.5 (C11), 69 (C12), and 68.7 (C13) ppmbelong to the cyclopentadienyl group.

The most frequently used supports in biotechnology for covalentenzyme immobilization have been obtained after activation of nat-ural and synthetic polymers such as chitin, alginic acid, cellulose,acrylic polymer and polyvinylalcohol [31–35]. Pyx/PyCO2Hy/PyFcz

having different monomer ratios were prepared from Py, Py-Fc andPyCO2H monomers as described in Section 2. The present methodwas effective in that the reactive carboxylic acid group was read-ily introduced into polymer support without any modification. Inaddition, the required functional group composition could also befixed by changing the co monomer ratio in the polymer preparationmixture. The carboxylic acid groups can bind the protein moleculesvia their amine groups at the pH range where the enzyme is sta-ble and does not lose activity by using CMC as a coupling agents(Fig. 1B). The amide bond formed between carboxylic acid groupand biomolecule is stable, so that carboxylic acid group contain-ing supports are useful for enzyme immobilization. The increaseof the composition of the PyCO2H increase the amount of boundGOx on the electrode as given in Table 1. The maximum enzymebinding was observed in the Py0.2/PyCO2H0.6/PyFc0.2 and the low-est was Py0.6/PyCO2H0.2/PyFc0.2. As a consequence, the catalyticcurrent response was not directly related to the amount of boundenzyme on the electrode and the Py0.2/PyCO2H0.2/PyFc0.6 coatedelectrode showed maximum amperometric response.

3.3. Electrocatalytic behavior of Py/Py-CO2H/Py-Fc mediated GOxelectrode

Amperometric enzyme-based biosensors are very suitable forthe monitoring of blood glucose level. The enzyme based glu-cose biosensors are commonly based on the immobilization ofglucose oxidase on working electrode used as transducer. Theliberation of hydrogen peroxide in the enzymatic reaction canbe measured amperometrically at the working electrode surface.However, there are two limitations which have hampered the rou-tine use of that system. The first limitation of the amperometricmonitoring of hydrogen peroxide which is produced enzymati-cally by GOx is the requirement of high measurement potentialwhere unwanted reactions of electroactive species in biologicalfluids. The second limitation is the restricted solubility of oxygenin biological fluids that limits the enzymatic reaction. In recentyears, the improvements were achieved by replacing oxygen witha non-physiological electron acceptor, which able to shuttle elec-tron from flavin redox center of the enzyme to the surface of theworking electrode. Using redox mediators, the measurement havebecome insensitive to oxygen fluctuations and can be carried outby applying lower potentials. The reaction mechanism of the medi-ated GOx based glucose biosensor can be summarized as follows[27,36]:

�-d-glucose + GOD (FAD) → GOD (FADH2)

+ d-glucono-�-lactone

GOD (FADH2) + 2Medox → GOD (FAD) + 2H+ + 2Medred

2Medred → 2Medox + 2e− (atelectrode)

M. S enel / Synthetic Metals 161 (2011) 1861– 1868 1865

of Py0

wvbIoc

F(p

Fig. 4. 1H and 13C NMR spectra

The electrocatalytic behaviors of GOx immobilized electrodeere evaluated by cyclic voltammetry. Fig. 5 shows the cyclic

oltammograms of the modified electrode in pH 7.0 phosphateuffer saline (PBS) (a) in the absence and (b) presence of glucose.

n the absence of glucose, only the cyclic voltammogram of Fc wasbserved. When glucose solution was added into the electrochemi-al cell, an electrocatalytic characteristic appeared with an increase

ig. 5. Cyclic voltammograms of the enzyme electrode of Py0.2/PyCO2H0.2/PyFc0.6

a) in the absence and (b) presence of 1.5 mM glucose at a scan rate of 25 mV/s inH 7.0 0.01 M PBS solution.

.4/PyCO2H0.4/PyFc0.2 copolymer.

of the decrease of the oxidation current. These results strongly indi-cate the enzyme-dependent catalytic oxidation of glucose, whichoriginated from the GOx reaction mediated by a polymeric medi-ator. A new polymeric mediator could effectively shuttle electronsfrom the electrode surface to the redox center of GOx.

3.4. Catalytic current for GOx electrode

Fig. 7 shows the steady-state catalytic current responses ofthe various enzyme electrodes carrying Py/PyCO2H/PyFc films asa function of glucose concentration at +0.38 V vs. Ag/AgCl. Thecatalytic current of the enzyme electrodes rises with increas-ing glucose concentration. It is likely that this behavior followsMichaelis–Menten kinetics, as observed for many amperometricenzyme electrodes [15,37,38].

Fig. 6 and Table 1 show that the catalytic current responseof the enzyme electrode changes with the copolymer composi-tion due to the conductivity changes in the copolymer films. Thecatalytic response of the enzyme electrode reaches its maximumat a Py0.2/PyCO2H0.2/PyFc0.6. Raising the concentration of the Fcunits increases the oxidation concentration near GOx. Reducedhydrophilicity of the copolymers might lead to reduced penetra-tion of glucose into the enzyme electrodes, which could lead toa fall in the catalytic current [15]. The amounts of bound GOx onthe copolymer film are directly related to the composition of thePy-CO2H units of copolymer (Table 1). Consequently, the catalytic

current response was not directly related to the amount of enzymebound to the electrode. The number of ferrocene units in the copoly-mer film was the deciding factor in the catalytic current responsein this system.

1866 M. S enel / Synthetic Metals 161 (2011) 1861– 1868

Fw

3

absr7d

fowtrl

3

Pbgr1iabat

ottrt

wss

Fig. 7. (A) Effect of the pH on the current response of enzyme electrode ofPy0.2/PyCO2H0.2/PyFc0.6 to glucose at an applied potential +0.38 V; (B) effect of tem-

matic biosensor in storage conditions (phosphate buffer of pH 7.5

ig. 6. Influence of copolymer ratios on amperometric response to glucose. Currentas measured at +0.38 V vs. Ag/AgCl after applying the potential for 100 s.

.5. Optimum pH and temperature

The activity of the enzymes depends on the pH of the mediumnd optimum pH of the enzyme may be effected from the immo-ilization method. The effect of pH on the enzyme electrode wastudied between 5.0 and 9.0. As shown in Fig. 7A, the currentesponse increased from pH 5.0 to 7.5, and decreased from pH.5 to 9.0. Therefore, pH 7.5 was used in further experiments andetermination of glucose.

The temperature of the medium is also an important parameteror the enzyme activity. The effect of temperature on the amper-metric response was also investigated in the range of 20–60 ◦C,hich is given in Fig. 7B. The response increased with increasing

he temperature and reached a maximum at 45 ◦C, and then theesponse decreased. This might be due to the denaturation or theeaching of the immobilized enzyme.

.6. Constant potential response of GOx electrode to glucose

The amperometric determination of glucose at they/PyCO2H/PyFc electrode has been investigated and the cali-ration curve of the response current of the GOx electrode tolucose concentration is shown in Fig. 8. The inset plot shows theesponse current of the electrode to the successive addition of

mM glucose. From Fig. 8, it can be observed that the linear ranges up to 5 mM with a correlation coefficient (R) of 0.9920 and then

plateau is reached gradually at higher glucose concentration. Theiosensor has a good detection limit of 6.9 �M (signal-to-noise = 3),

high sensitivity of 1.796 �A mM−1 cm−2, and a short responseime (within 2 s).

The apparent Michaelis–Menten constant Kmapp is an indicator

f the enzyme-substrate reaction kinetics and is used to evaluatehe biological activity of the immobilized enzyme. According tohe Lineweaver–Burk form of the Michaelis–Menten equation, theelationship between the response current and the glucose concen-ration is obtained as follows:

1Iss

= Kmapp

ImaxC+ 1

Imax

here Iss is the steady-state response current after the addition ofubstrate; Imax is the maximum response current under saturatedubstrate conditions; and C is the bulk concentration of glucose.

perature on the current response of enzyme electrode of Py0.2/PyCO2H0.2/PyFc0.6 toglucose in 10 mM PBS solution, pH 7.5 at an applied potential of +0.38 V vs. Ag/AgCl.

The apparent Michaelis–Menten constant Kmapp in the present

study is calculated to be 4.73 mM, lower than that reported in lit-erature [39], which indicates that the enzyme immobilized on theelectrode maintains its bioactivity. In Table 2 the analytical perfor-mance of this biosensor is compared with that of the prior studies.The analytical characteristics of the enzyme electrode indicate thatthe enzyme was effectively attached into the conducting polymerfilm and that the bioactivity of the immobilized enzyme was wellpreserved in the enzyme electrode.

3.7. Reproducibility, stability and interference studies

The reproducibility of five GOx immobilized electrodes was esti-mated by the response to 1 mM glucose at the potential of +0.38 V.The results reveal that the sensor has satisfied reproducibilitywith a relative standard deviation of 7.4%. The stability of enzy-

at 4 ◦C) was investigated in the same phosphate buffer solutioncontaining 1.0 mM glucose. The corresponding result shows that82% response current is still retained after 28 days. This result

M. S enel / Synthetic Metals 161 (2011) 1861– 1868 1867

Table 2Comparison of the analytical performances of the glucose biosensors.

Electrode RT (s) Linear range Detection limit Sensitivity Ref.

Py0.2/PyCO2H0.2/PyFc0.6 2 1.0–4.0 mM 6.9 �M 1.796 �A mM−1 cm−2 This workTh/ThCO2H /ThFc 4 0.5–3.0 mM 2.5 �M 40 nA mM−1 cm−2 [15]PyCO2H – 1.0–80 mM – 0.85 �A mM−1 cm−2 [40]Py-Fc 4 1.0–18 mM – 0.05 �A mM−1 cm−2 [21]Py 15 0–5.0 mM – 80 nA mM−1 [41]Py – 1.0–22 mM – 271 nA mM−1 [42]

RT, response time; Py, poly(pyrrole); PyCO2H, poly (3-(1H-pyrrol-1-yl)propanoic acid); Ppoly (3-thiophene acetic acid); ThFc, dicyclopentadienyl iron-1,4-dienylmethyl-2-(thioph

Fig. 8. Calibration curve of the amperometric response of the enzyme electrode ofPy0.2/PyCO2H0.2/PyFc0.6 to glucose concentration in 0.01 M PBS. The inset plot showsta

ibsm

r0Tpisn

4

ttPohfahiibs

[[[

[[[[[

[

[

[[[[[

[[

[[[

[tions, 2nd ed., Wiley, New York, 2001.

[31] S. Akgöl, Y. Kacar, A. Denizli, M.Y. Arıca, Food Chem. 74 (2001) 281.[32] K. Sarıoglu, N. Demir, J. Acar, M. Mutlu, J. Food Eng. 47 (2001) 271.[33] G. Spagna, R.N. Barbagallo, P.G. Pifferi, R.M. Blanco, J.M. Guisan, J. Mol. Catal. B

he current response of the enzyme electrode to successive glucose injections at anpplied potential +0.38 V in stirred 0.1 M PBS.

mplies that the GOx immobilized electrode is considerably sta-le. Good stability may be attributed to the enzyme attachedtrongly on the Py0.2/PyCO2H0.2/PyFc0.6 film that is stable in theedium.The effect of the interferents was studied on the amperomet-

ic response of the enzyme electrode by three main interferents:.1 mM uric acid, 0.1 mM ascorbic acid and 0.3 mM acetaminophen.hese substances were added into the reaction mixture in theresence of 2 mM glucose. It was found that the presence of the

nterferents had a relative error of less than 5% in the current mea-urement, therefore, this enzyme electrode can detect glucose withegligible interferences.

. Conclusions

A novel ferrocene-functionalized poly-pyrrole was prepared forhe development of an amperometric biosensor for glucose detec-ion. This biosensor can be prepared by electropolymerization ofy, Py-CO2H, and Py-Fc monomers on GCE at a constant potentialf +1.2 V. We have demonstrated that the conducting poly-pyrroleaving acid and ferrocene groups can be utilized as a suitable matrix

or the covalent attachment of enzyme through peptide linkagend polymeric redox mediator. The catalytic current response wasighly sensitive and revealed a near-linear increase with increas-

ng glucose concentration up to 5.0 mM. The methods described forntroducing the electron-mediating groups in the present study wille available for practical fabrication of the enzyme electrodes con-isting of Py/Py-CO2H/Py-Fc copolymer and various redox enzymes.

[

[[

yFc, N-(3-(1H-pyrrol-1-yl)ethyl)ferrocenecarboxate; Th, poly(thiophene); ThCO2H,en-3-yl)acetate.

This new glucose enzyme electrode shows a fast, sensitive responseto glucose and is relatively inexpensive. This copolymer might pro-vide a useful platform for incorporation of other enzymes andsubstrate couples during the construction of biosensors for a varietyof bioanalyses.

Acknowledgment

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

References

[1] A. Heller, Acc. Chem. Res. 23 (1990) 128.[2] S.C. Barton, J. Gallaway, P. Atanassov, Chem. Rev. 104 (2004) 4867.[3] X.Q. Cui, C.M. Li, J.F. Zang, S.C. Yu, Biosens. Bioelectron. 22 (2007) 3288.[4] J.M. Liu, C. Guo, C.M. Li, Y. Li, Y. Li, Q. Chi, X. Huang, Electrochem. Commun. 11

(2009) 202.[5] X.J. Zhang, H.X. Ju, J. Wang, Electrochemical Sensors, Biosensors and their

Biomedical Applications, Elsevier, New York, 2008.[6] S. Cosnier, Biosens. Bioelectron. 14 (1999) 443.[7] F. Garnier, H. Korri-Youssoufi, P. Srivastava, A. Yassar, J. Am. Chem. Soc. 116

(1994) 8813.[8] J. Roncali, J. Chem. Rev. 92 (1992) 711.[9] H. Korri-Youssoufi, B. Makrouf, A. Yassar, Mater. Sci. Eng. C 15 (2001) 265.10] T. Saito, M. Watanabe, React. Funct. Polym. 37 (1998) 263.11] S. Koide, K. Yokoyama, J. Elect. Anal. Chem. 468 (1999) 193.12] N. Kuramoto, Y. Shishido, K. Nagai, Macromol. Rap. Commun. 15 (1994)

441.13] N. Kuramoto, Y. Shishido, Polymer 3 (1998) 669.14] M. Senel, E. Cevik, M.F. Abasiyanik, Sens. Actuators B 145 (2010) 444.15] M.F. Abasiyanik, M. Senel, J. Electroanal. Chem. 639 (2010) 21.16] S.H. Lee, H.Y. Fang, W.C. Chen, Sens. Actuators B 117 (2006) 236.17] B.R. Eggins, Chemical Sensors and Biosensors, John Wiley and Sons Ltd., Eng-

land, 2002, p. 154.18] A.E.G. Cass, G. Davis, G.D. Francis, H.A.O. Hill, W.J. Aston, I.J. Higgins, E.V. Plotkin,

L.D.L. Scott, A.P.F. Turner, Anal. Chem. 56 (1984) 667.19] J.M. Dicks, W.J. Aston, G. Davis, A.P.F. Turner, Anal. Chim. Acta 182 (1986)

103.20] J. Wang, L. Wu, W. Lu, R. Li, J. Sanchez, Anal. Chim. Acta 228 (1990) 251.21] N.C. Foulds, C. Lowe, Anal. Chem. 60 (1988) 2473.22] A. Mulchandani, C.L. Wang, H.H. Weetall, Anal. Chem. 67 (1995) 94.23] M. Marchesiello, E.M. Geniés, Electrochim. Acta 37 (1992) 1987.24] K. Abu-Rabeah, I.I. Nita, A.M. Tencaliec, R.S. Marks, Electrochim. Acta 54 (2009)

4359.25] K. Abu-Rabeah, R.S. Marks, Sens. Actuators B: Chem. 136 (2009) 516.26] E. Saint-Aman, M. Ungureanu, T. Visan, J.-C. Moutet, Electrochim. Acta 42 (1997)

1829.27] W. Schuhmann, Biosens. Bioelectron. 10 (1995) 181.28] S.E. Wolowacz, B.F.Y. Yon-Hin, C.R. Lowe, Anal. Chem. 64 (1992) 1541.29] O. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951)

263.30] A.J. Bard, L.R. Faulkner, Electrochemical Methods: Fundamentals and Applica-

11 (2000) 63.34] E. Yapar, S.K. Kayahan, A. Bozkurt, L. Toppare, Carbohydr. Polym. 76 (2009)

430.35] M. S enel, A. C oskun, M.F. Abasıyanık, A. Bozkurt, Chem. Papers 64 (2010) 1.36] A. Chaubey, B.D. Malhotra, Biosens. Bioelectron. 17 (2002) 441.

1 etals 1

[

[[

868 M. S enel / Synthetic M

37] J.D. Qiu, W.M. Zhou, J. Guo, R. Wang, R.P. Liang, Anal. Biochem. 385 (2009)264.

38] J.D. Qui, M.Q. Deng, R.P. Liang, M. Xiong, Sens. Actuators B 135 (2008) 181.39] J. Li, X.Q. Lin, Biosens. Bioelectron. 22 (2007) 2898.

[[[

61 (2011) 1861– 1868

40] K. Kojima, T. Yamauchi, M. Shimomure, S. Miyauchi, Polymer 39 (1998) 2079.41] W.J. Sung, Y.H. Bae, Biosens. Bioelectron. 18 (2003) 1231.42] A. Guerrieri, G.E. De Benedetto, F. Palmisano, P.G. Zambonin, Biosens. Bioelec-

tron. 13 (1998) 103–112.