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Current Applied Physics 12 (2012) 1158e1165

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Current Applied Physics

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A novel amperometric hydrogen peroxide biosensor based on pyrrole-PAMAMdendrimer modified gold electrode

Mehmet Senel a,*, Emre Çevik b

aDepartment of Chemistry, Faculty of Arts and Sciences, Fatih University, 34500 B.Cekmece, Istanbul, TurkeybDepartment of Genetics and Bioengineering, Faculty of Engineering, Fatih University, 34500 B.Cekmece, Istanbul,Turkey

a r t i c l e i n f o

Article history:Received 17 October 2011Received in revised form6 February 2012Accepted 10 February 2012Available online 17 February 2012

Keywords:DendrimerPyrrolePAMAMHorseradish peroxidaseBiosensorCovalent immobilization

* Corresponding author. Department of Chemistr34500 B.Cekmece, Istanbul, Turkey. Tel.: þ90 22128663402.

E-mail address: msenel@fatih.edu.tr (M. Senel).

1567-1739/$ e see front matter � 2012 Elsevier B.V.doi:10.1016/j.cap.2012.02.040

a b s t r a c t

Horseradish peroxidase (HRP) was immobilized into an electrochemically prepared copolymer ofpyrroleePAMAM (PAMAM; polyamidoamine) dendrimers for the construction of amperometrichydrogen peroxide biosensor. First, second, and third generation amidoamineepyrrole dendrons havingbranched amine periphery and focal pyrrole functionality were synthesized via divergent pathway.Pyrrole dendrimers were covalently attached onto the electrode surface and polymerized by electro-chemical copolymerization with pyrrole monomer. The synthesized dendrimers and copolymers havebeen characterized by FTIR-ATR and NMR. These copolymers have been utilized as conducting films foramperometric hydrogen peroxide sensing. The HRP retains its bioactivity after immobilization into thedendronized pyrrole-copolymers. Amperometric response was measured as a function of concentrationof hydrogen peroxide, at fixed potential of þ0.35 V vs. Ag/AgCl in a phosphate buffered saline (pH 7.5).The effect of pH and temperature of the medium, storage, and reusability properties were investigated.The results indicate an efficient immobilization of enzyme onto the PAMAM type dendrimer modifiedsurface containing pyrrole monomer, which leads to high enzyme loading, and increased lifetimestability of the electrode.

� 2012 Elsevier B.V. All rights reserved.

1. Introduction

The development of biosensors requires the achievement of anefficient interface between the biomolecules and the electronictransducers. Conducting polymer interfaces are particularly suit-able for localizing biomolecules onto surfaces [1]. The poly-conjugated conducting polymers were recently proposed forbiosensing applications because of number of favorable character-istics such as (1) direct and easy deposition on the electrodesurface, (2) control of thickness, and (3) redox conductivity of andpolyelectrolyte characteristics of polymer useful for sensor appli-cation [2]. Polypyrrole fulfills the above requirements together withhaving the characteristics of easy oxidation, high chemical stability,and low cost of monomer [3].

Dendrimers have extensively been studied in the past threedecades due to the unique architectural and functional controlachieved during their synthesis [4e6]. Despite such an activity,

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enriching the structural diversities of dendrimers, researches havefocused on dendrimers with small cores, until a breakthrough inthe synthesis of dendronized polymers was reported [7] thatrecognized the significance of dendron functionalization for thebackbone conformation and the overall shape of the resultingmacromolecules. Recently, there has been an increasing interest toexploit the dendritic macromolecules or dendron appendages forchemical and surface modification of silica [7], carbon [8], chitosan[9], DNA [10], and thiophene [11]. Among various dendrimers,poly(amidoamine) (PAMAM) dendrimers are the most frequentlystudied. In recent years, some reports have shown that the PAMAMdendrimers can be used as the bioconjugating reagents forconstruction of glucose oxidase electrode [12,13]. In another study,acetylcholinesterase and choline oxidase were co-immobilizedonto the Au electrode by means of PAMAM dendrimer for thepesticide analysis [14]. Use of PAMAM for the immobilization ofalcohol dehydrogenase (ADH) onto carbon cloth platforms and itsapplication as bioanode in ethanol/O2 biofuel cells was carried outby Forti et al. [15]. Additionally, horseradish peroxidase (HRP),glucose oxidase (GOx), pyranose oxidase (PyOx), alcohol oxidase(AOx) and fructose dehydrogenase (FDH) were immobilized on toAu gold surface modified by PAMAM/cysteamine [13,16e18].

M. Senel, E. Çevik / Current Applied Physics 12 (2012) 1158e1165 1159

The determination of hydrogen peroxide is of great importancein textile/food industries and health care. Many techniques,including titrimetry [19], chemiluminescence [20], fluorescence[21], and spectrophotometry [22] tend to be complex, time-consuming, and suffer from various interferences. Consequently,rapid and reliable methods for measuring H2O2 are naturallyrequired. Electrochemical methods, especially various ampero-metric H2O2 biosensors, have been extensively employed for thedetermination of H2O2. Several valuable efforts have been focusedon developing electrochemical biosensors of horseradish peroxi-dase for the determination of H2O2. Horseradish peroxidase (HRP)is one of the heme-containing redox enzymes. The electron transferconverts Fe(III) in the heme of HRP to Fe(II). This process cancatalyze some chemical reaction such as the reduction of H2O2 [23].The performance of these biosensors depends on the methods ofenzyme immobilization when fabricating enzyme-modified elec-trodes. Various immobilization techniques andmaterials have beenemployed, including adsorption [24], cross-linking [25], layer-by-layer assembly [26], covalent binding [27], surfactanteenzymecomplexation [28], solegel entrapment [29], and use of biologicalmembranes [30] and nanomaterials [31], and so on. As initiallysynthesized, PAMAM dendrimers have an amine (eNH2)-cappedsurface. They can also be terminated with a carboxylic-acid-capped surface (eCOOH) or an acetamide-capped surface(eNHC(O)CH3) [32].

The present paper reports the results on the use ofpyrroleePAMAM dendritic wedges for preparation of conductingpolypyrrole having dendritic substituent to obtain conductingpolymer-modified electrode. To the best of our knowledge, this isthe first example of the use of dendronized-conducting polymer forthe development of enzyme biosensor. We have recently useddendrimers as proton-conducting membranes [33] as well as pol-ychelatogens [34]. First, second, and third generationamidoamineepyrrole dendrons having branched amine peripheryand focal pyrrole functionality were synthesized via divergentpathway. Polypyrrole dendrimers were synthesized using electro-chemical copolymerization of amidoamineepyrrole dendrons withpyrrole monomer. The synthesized dendrimers and copolymershave been by FTIR-ATR and NMR. These copolymers have beenutilized as conducting films for enzymatic amperometric H2O2sensing. The advantage of using dendritic pyrrole modified surfacefor efficient enzyme immobilization and the relationship betweengeneration of dendrimers and sensor properties were described.

2. Experimental

2.1. Materials

HRP (EC 1.11.1.7, RZ >3.0, 250 U/mg), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and N-hydroxy-succinimide (NHS) were obtained from Sigma ChemicalCo. 1-Cyanoethylpyrrole monomer was obtained from Aldrich.Pyrrole monomer, p-toluene sulphonic acid sodium salt, and H2O2were procured from Fluka. Methyl acrylate and ethylenediaminewere obtained from Merck. All other chemicals were of analyticalgrade and were used without further purification.

2.2. Synthesis of N-(3-aminopropyl)-pyrrole

A solution of 1-(2-cyanoethyl)pyrrole (0.02 mol) in anhydrousether (15 mL) was added dropwise to a suspension of LiAlH4(0.05 mol) in anhydrous ether (150 mL), and the mixture wasrefluxed for 10 h. After cooling, the excess hydridewas destroyed bysuccessive addition of water (1.7 mL), a solution of 15% (w/v) NaOH(1.7 mL), and water (5.1 mL). The solution was heated to 40 �C for

2 h and filtered on Celite before evaporating to dryness. A yellow oilwas obtained with a yield of 91.6%. 1H NMR d (CDCl3): 1.90 (m, 2H,CH2-2), 2.70 (t, 2H, CH2-3), 3.95 (t, 2H, CH2-1), 6.14 (d, 2H, CH-b),6.65 (d, 2H, CH-a). 13C NMR d (CDCl3): 35.5 (CH2-2), 39.7 (CH2-3).FTIR spectroscopy: the characteristic features of the N-(3-aminopropyl)pyrrole spectrum are a strong sharp peak at3370 cm�1 with a medium shoulder at 3295 cm�1 that correspondsto the “free” asymmetrical and symmetrical NeH stretchingvibration modes of the aliphatic primary amines.

2.3. Synthesis of pyrroleePAMAM dendritic wedges (G1, G2, G3)

Divergent synthesis (Fig. 1) of the amine-terminatedpyrroleePAMAM dendrimer was carried out by initial Michaeladdition of methanolic solution of N-(3-aminopropyl)-pyrrole withexcess methyl acrylate (1:10 molar ratio). The reaction mixture wasstirred for three days at room temperature. The excess methylacrylate was removed under vacuum at 40e50 �C temperature toafford the ester-functionalized derivative G0.5. The reactionmixture was next submitted to the reaction sequence leading to thenext generation pyrroleePAMAM dendrimer G1.5, consisting of theexhaustive amidation of the ester-functionalized G0.5 to ethyl-enediamine (1:30 molar ratio), followed by Michael addition of theresulting amine with methyl acrylate (20 equiv of G0.5). Excessreagents were removed under vacuum at 60e70 �C temperature.Repetition of this two-step procedure ultimately leads to the nextgeneration pyrroleePAMAM dendrimer G3. The dendrimers G2 andG3, isolated in 85e90% yield, were gummy in nature.

2.4. Preparation of enzyme electrodes

The preparation of the enzyme electrode is shown in Fig. 2.Before each new measurements, the gold electrode was polishedwith 1.0, 0.5, and 0.3 mm alumina slurry, sonicated consequentiallyin distilled water and absolute ethanol for 15 min each and etchedfinally in 0.5 mol L�1 H2SO4 solution by cyclic-potential scanningbetween �0.3 and þ1.7 V until a reproducible voltammetricresponse was obtained.

The cleaned gold electrode was first immersed in 20 mmol L�1

3-mercaptopropionic acid (MPA) aqueous solution for 2 h. After theelectrode was thoroughly rinsed with water to remove physicallyadsorbed MPA. PyePAMAM dendrimers were linked chemically tothe functionalized gold electrodes by promoting the creation ofamide bonds NH(CO) between the COOH ends of the MPA andamine peripheral groups (NH2) of the PyePAMAMdendrimers. Thiswas achieved by immersing the thiolated gold electrodes inmethanolic solutions containing 5�10�3 mol L�1 EDC (couplingreagent to create amide bonds) and 21�10�6 mol L�1 PyePAMAMdendrimers of generation 1.0 (G1), 2.0 (G2) and 3.0 (G3) for 12 h atRT [35]. Subsequently, the dendrimer-functionalized gold surfaceswere washed by dipping them in gently stirred MeOH at roomtemperature.

Finally, the HRP enzyme was immobilized on the modifiedelectrode by electropolymerization of pyrrole on the modifiedelectrode from aqueous solution containing 5 mgmL�1 HRP,50 mM pyrrole and 1.0 M p-toluene sulphonic acid sodium salt ata fixed voltage of 0.8 V vs. Ag/AgCl. The resulting electrode waswashed with PBS (pH 7.0) and stored in the same PBS at 4 �C whennot in use.

2.5. Apparatus

The IR absorption spectra (4000e400 cm�1) were recordedwitha Mattson Genesis II spectrophotometer. NMR spectra were recor-ded in CDCl3 using a Bruker 400 MHz spectrometer.

Fig. 1. Synthetic pathway of pyrroleePAMAM dendrimers i) methyl acrylate in methanol, ii) ethylenediamine in methanol, iii) methyl acrylate in methanol, iv) ethylenediamine inmethanol, v) methyl acrylate in methanol, vi) ethylenediamine in methanol.

Fig. 2. Scheme of preparation process of the HRP biosensor.

M. Senel, E. Çevik / Current Applied Physics 12 (2012) 1158e11651160

Fig. 3. FTIR spectra of PyePAMAM dendrimers with different generations.

M. Senel, E. Çevik / Current Applied Physics 12 (2012) 1158e1165 1161

Electrochemical measurements were performed using a CHIModel 842B electrochemical analyzer. A small gold working elec-trode (1 cm�2), a platinum electrode (1 cm�2), an Ag/AgCl-saturated KCl reference electrode and a conventional three-electrode electrochemical cell were purchased from CHInstruments.

All amperometric measurements were carried out at roomtemperature in stirred solutions by applying the desired potentialand allowing the steady-state current to be reached. Onceprepared, the HRP electrodes were immersed in 10 ml pH 7.410 mMPBS solution and the amperometric response to the additionof a known amount of H2O2 solutionwas recorded. The data shownare the average of three measurements for each electrode.

Fig. 4. 1H NMR spectrum of Py

The morphology of the modified gold surfaces was imaged byatomic force microscopy (AFM, Park systems XE-100E). Imagingwas carried out at ambient temperature in noncontact mode.

3. Results and discussion

3.1. Characterization of pyrroleePAMAM dendritic wedges andmodified electrodes

Fig. 3 shows a comparison of the FTIR spectra between 4000 and400 cm�1 of the PyePAMAM dendrimers from G0 to G3. The peaksaround at 3280 cm�1 can be attributed to the stretching vibration ofNeH, indicating the growing of the PAMAM dendrimers on the Pymonomer, other characteristic bands at around 1640 cm�1 areassigned to the vibration of the amide group in PAMAM den-drimers. Moreover, the intensity of these bands at 3280 and1640 cm�1 obviously increased with increasing the generation ofthe dendrimers.

The structure of PyePAMAM dendrimer (G3) can be determinedfrom the detailed 1H NMR spectrum (Fig. 4). In the spectrum, thepeaks between 6.1 and 6.6 ppm are specific resonance signals ofpyrrole ring protons. The peaks between 2.0 to 3.4 ppm areattributed to the growing of the PAMAM dendrimers on pyrrolemonomer, respectively.

Cyclic voltammetry of ferrocyanide, as redox marker, is a valu-able and useful technique to follow the barrier of the modifiedelectrode. Cyclic voltammograms of 5 mM [Fe(CN)6]3�/4�

between�0.2 and 0.5 V shown in Fig. 5Awere obtained at differentmodified electrodes; (a) bare Au electrode, (b) Au/MPA, (c) Au/MPA/PyeNH2eG0, (d) Au/MPA/PyePAMAMeG1, (e) Au/MPA/PyePAMAMeG2, and (f) Au/MPA/PyePAMAMeG3, respectively.Well-defined cyclic voltammogram, characteristic of a diffusion-controlled redox process, is observed at the bare Au electrode(Fig. 5Aa). After themodification of the Au electrode by dipping intoMPA solution, and the covalent attachment of the PyePAMAMdendritic wedges, the well-defined peaks of the bare electrodewere greatly diminished, confirming that the surface of the Au

ePAMAM dendrimer G3.

M. Senel, E. Çevik / Current Applied Physics 12 (2012) 1158e11651162

electrode was successfully modified. As can be seen in Fig. 5A, theCV responses of ferrocyanide solution on the bare Au and the MPA-modified electrode are different compared to those obtained usingthe PyePAMAM dendrimers-modified electrodes. Consequently,the electron transfer rate between the ferrocyanide solution andthe electrode surface is strongly dependent on the dendrimergeneration [35]. Fig. 5B shows the cyclic voltammograms of thepolypyrrole film growth during the electrolysis of the solutioncontaining 50 mM pyrrole and 1.0 M p-toluene sulphonic acidsodium salt in phosphate buffer with pH 7.5. The current begins toincrease markedly at around 0.85 V, and then increases continu-ously with increasing potential.

3.2. Optimization of the experimental parameters

The effect of applied potential on the amperometric response ofthe biosensor was evaluated in the range ofþ0.1 toþ0.5 V (Fig. 6A).The response approached a plateau value at þ0.35 mV, which was

Fig. 5. (A) Cyclic voltammograms of a) bare Au electrode, b) Au/MPA, c) Au/MPA/PyeNH2eG0, d) Au/MPA/PyePAMAMeG1, e) Au/MPA/PyePAMAMeG2 and f) Au/MPA/PyePAMAMeG3 electrodes in solution 10 mM PBS solution containing 5 mM[Fe(CN)6]3�/4�. Scan rate is 50 mV/s; (B) cyclic voltammogram of polypyrrole filmgrowth on the Au/MPA/PyePAMAMeG3 electrode from the solution containing5 mgmL�1 HRP, 50 mM pyrrole and 1.0 M p-toluene sulphonic acid sodium salt at thescan rate 25 mV/s.

selected as the working potential for amperometric determinationsof H2O2.

The pH of the medium is an important parameter for theenzyme activity. The effect of pH on the enzyme electrode was

Fig. 6. (A) Effect of the potential on the current response of Au/MPA/PyePAMAMeG2/HRP to 5 mM H2O2 in pH 7.5 10 mM PBS solution; (B) effect of the pH on the currentresponse of Au/MPA/PyePAMAMeG2/HRP to 5 mM H2O2 at an appliedpotential þ0.35 V; (C) effect of temperature on the amperometric response of 5 mMH2O2 in 10 mM PBS solution, pH 7.5 at an applied potential of þ0.35 V vs. Ag/AgCl.

Fig. 7. Dependence of current on H2O2 concentration for enzyme electrodes at þ0.35 V vs. Ag/AgCl for (A) Au/MPA/PyeNH2/HRP; (B) Au/MPA/PyePAMAM-G1/HRP; (C) Au/MPA/PyePAMAMeG2/HRP; (D) Au/MPA/PyePAMAMeG3/HRP electrodes. Insets show the linear calibration curve.

Table 1Comparison of the analytical performance of the different modified electrodes.

Electrode RT(s)

LR(mM)

DL(mM)

Sensitivity(nA/mM)

KMapp

(mM)

Au/MPA/PyeNH2/HRP 4 1e14 10.8 12 40.7Au/MPA/PyePAMAMeG1/HRP 2 1e8 14.6 24 9.5Au/MPA/PyePAMAMeG2/HRP 3 1e18 5.7 54 5.8Au/MPA/PyePAMAMeG3/HRP 6 1e10 9.3 17 24

Au, gold electrode; RT, response time; LR, linear range; DL, detection limit.

M. Senel, E. Çevik / Current Applied Physics 12 (2012) 1158e1165 1163

studied between 5.0 and 9.0. As shown in Fig. 6B, the currentresponse increased from pH 5 to 7.5, and decreased from pH 7.5 to9.0, which is in agreement with that reported earlier [36]. There-fore, pH 7.5 was used in further experiments and determination ofH2O2.

The effect of temperature on the amperometric response wasalso investigated in the range of 30e55 �C, which is given in Fig. 6C.The response increased with increasing the temperature andreached a maximum at 40 �C, and then the response decreased.This might be due to the denaturation or the leaching of theimmobilized enzyme.

3.3. Amperometric current response of enzyme electrodes

The steady-state catalytic current response of the variousenzyme electrodes is shown as a function of hydrogen peroxideconcentration at þ0.35 V vs. Ag/AgCl in Fig. 7. The analyticalperformances of the different modified electrodes are summarizedin Table 1. On the base of the comparative study between the abovefour modified electrodes, it can be seen that the proposedbiosensor, that is, the Au/MPA/PyePAMAMeG2/HRP, exhibiteda broader linear range and lower detection limit than the others.Especially, the sensitivity of 54 nA/mMcm2 obtained with the Au/MPA/PyePAMAMeG2/HRP was 4.5 times higher than Au/MPA/PyeNH2/HRP. This value was 2.3 times higher than Au/MPA/PyePAMAMeG1/HRP, and 3.2 times higher than Au/MPA/PyePAMAMeG3/HRP. This fact demonstrated clearly that

PyePAMAMeG2 dendritic wedge as an immobilization platformfor enzymes was superior to PyeNH2.

As shown in the inset of Fig. 7C, when the concentration ofhydrogen peroxide was higher than 9 mM, the response of theenzyme electrode deviated from the straight line, reaching plateau,showing that the behavior follows MichaeliseMenten kinetics, asseen for many amperometric enzyme electrodes [37]. In Table 2 theanalytical performance of this biosensor is compared with that ofthe prior studies. The strong adsorption of HRP on the electrodesurface could cause denaturation. On the other hand, the electro-chemically active centers in HRP are always buried deeply in itsextended three-dimensional structure, which makes direct elec-tron transfer between HRP and the electrode surface very difficult.Some materials, such as self-assembled monolayers [38] andsurfactants [39] were introduced to realize direct electron transferbetween HRP and electrode. In this study, polyamidoaminedendritic structure of copolymer provides a suitable environment

Table 2Comparison of the analytical performance of the H2O2 biosensor based on HRP.

Electrode RT(s)

Linear range Detectionlimit

Sensitivity Ref.

PyePAMAMeG2 3 1.0e18 mM 5.7 mM 54 nA/mMcm2 This workP(GMA-co-VFc) 4 2.0e30 mM 2.6 mM 10.42 nA/mMcm2 [37]P(m-AAMFc) e 0.08e15 mM 0.08 mM 34 nA/mM [41]MDMS e 0.2e680 mM 0.078 mM e [36]CSeTHEOS 5 1.0e250 mM 0.4 mM e [42]FBCS 20 35e2000 mM 15 mM e [43]Fe3O4/dextran 10 2� 10�2 to

0.68 mM0.078�10�6 M

e [36]

Ti/TiO2/Au 5 0.05 to 4�10�2 mM

2.0�10�6 M

1.75 mA/mM [44]

CS/CoFe2O4 4 3� 10�2 to 8 mM 2.0�10�6 M

23 nA/mM [45]

GCE, glassy carbon electrode; RT, response time; P(m-AAMFc), poly(m-aminoanilinomethylferrocene); CS, chitosan; THEOS, tetrakis(2-hydroxyethyl)orthosilicates; FBCS,ferrocene branched chitosan.

M. Senel, E. Çevik / Current Applied Physics 12 (2012) 1158e11651164

for HRP. In addition to that, the conducting polypyrrole backboneand peripheral amine groups of PAMAM dendrimers increaseelectron transfer rate. The apparent MichaeliseMenten constant(KM

app), which gives an indication of the enzymeesubstrate kinetics

Fig. 8. (A) Reusability of the HRP immobilized electrode. Each data point representsthe average of data collected by three electrodes (pH 7.5; w25 �C); (B) storagestabilities of HRP immobilized electrode. The amperometric responses of these enzymeelectrodes are regularly checked during 60 days (pH 7.5; w25 �C).

for the biosensor, is calculated to be 5.8 mM using theLineweavereBurk equation [40]. The low value of KM

app means thatthe enzyme immobilized in PyePAMAMeG2 retained its activityand that there was a low-diffusion barrier.

3.4. Stability and reusability

The reusability of HRP electrode was obtained by runningmeasurements in the same day. Between each subsequentmeasurement electrodes stored at 4 �C in the buffer solution for10 min. The first 11 measurements revealed the same response, anactivity loss of 35% was observed with the subsequent use (Fig. 8A).The response of the HRP electrode was measured of its response to5 mM H2O2 for a period of 60 days. As shown in Fig. 8B theamperometric response of the enzyme electrode remained constantfor 20 days, and then an activity loss of 20% was observed, so itretained about 80% of its initial enzyme activity up to 2months. Thisstability behavior of the immobilized enzyme onto the modifiedsurface is better than the recently reported H2O2 biosensor [28].

4. Conclusions

In this work, novel pyrroleePAMAM dendrimers were synthe-sized with different generations and used for the construction ofamperometric hydrogen peroxide biosensor. Prepared dendrimerswere covalently attached on the surface of the MPA-modified Auelectrode, and then HRP was immobilized on this new host byelectropolymerization of pyrrole. The experimental results clearlyexhibited that the immobilized HRP possesses excellent catalyticability and well-retained activity. The results indicate an efficientimmobilization of enzyme onto the PAMAM type dendrimermodified surface containing pyrrole monomer, which leads to highenzyme loading, and increased lifetime stability of the electrode.The present data describes that the pyrroleePAMAM dendriticwedges-modified electrodes can be useful in the bio-electrochemical studies.

References

[1] H.K. Youssoufi, B. Makrouf, A. Yassar, Mater. Sci. Eng. C 15 (2001) 265e268.[2] Rajesh, V. Bisht, W. Takashima, K. Kaneto, React. Funct. Polym. 62 (2005)

51e59.[3] F. Diaz, J.I. Castillo, J.A. Logan, W.Y. Lee, J. Electroanal. Chem. 129 (1981)

115e132.[4] D.A. Tomalia, Aldrichimica Acta 37 (2004) 39e41.[5] S.M. Grayson, J.M.J. Frechet, Chem. Rev. 101 (2001) 3819e3868.[6] V.V. Narayanan, G.R. Newkome, Top. Curr. Chem. 197 (1998) 19e77.[7] R. Freudenberger, W. Claussen, A.D. Shluter, H. Wallmeier, Polymer 24 (1994)

4496e4501.[8] T. Gao, E.S. Tillman, N.S. Lewis, Chem. Mater. 17 (2005) 2904e2911.

M. Senel, E. Çevik / Current Applied Physics 12 (2012) 1158e1165 1165

[9] N. Tsubukowa, T. Takayama, React. Funct. Polym. 43 (2000) 341e350.[10] I. Goessl, L. Shu, A.D. Shluter, J.P. Rabe, J. Am. Chem. Soc. 124 (2002) 6860e6865.[11] P.R.L. Malenfant, J.M.J. Frechet, Macromolecules 33 (2000) 3634e3640.[12] H.C. Yoon, M.Y. Hong, H.S. Kim, Anal. Chem. 72 (2000) 4420e4427.[13] Z.M. Liu, Y. Yang, H. Wang, Y.L. Liu, G.L. Shen, R.Q. Yu, Sens. Actuators, B 106

(2005) 394e400.[14] M. Snejdarkova, L. Svobodova, G. Evtugyn, H. Budnikov, A. Karyakin,

D.P. Nikolelis, T. Hianik, Anal. Chim. Acta 514 (2004) 79e88.[15] J.C. Forti, S.A. Neto, V. Zucolotto, P. Ciancaglini, A.R. de Andrade, Biosens.

Bioelectron. 26 (2011) 2675e2679.[16] M. Akin, M. Yuksel, C. Geyik, D. Odaci, A. Bluma, T. Hopfner, S. Beutel,

T. Scheper, S. Timur, Biotechnol. Prog. 26 (2009) 896e906.[17] M. Akin, A. Prediger, M. Yuksel, T. Höpfner, D. Odaci Demirkol, S. Beutel,

S. Timur, T. Scheper, Biosens. Bioelectron. 26 (2011) 4532e4537.[18] K. Damar, D. Odaci Demirkol, Talanta 87 (2011) 67e73.[19] E.C. Hurdis, J.H. Romeyn, Anal. Chem. 26 (1954) 320e325.[20] S. Hanaoka, J.M. Lin, M. Yamada, Anal. Chim. Acta 426 (2001) 57e64.[21] L.S. Zhang, G.T.F. Wong, Talanta 48 (1999) 1031e1038.[22] C. Matsubara, N. Kawamoto, K. Takamura, Analyst 117 (1992) 1781e1784.[23] J.G. Zhao, R.W. Henkens, J. Stonehuerner, J.P.O. Daly, A.L. Crumbliss,

J. Electroanal. Chem. 327 (1992) 109e119.[24] Y. Xiao, H.X. Ju, H.Y. Chen, Anal. Biochem. 278 (2000) 22e28.[25] L. Qian, X. Yang, Talanta 68 (2006) 721e727.[26] P. He, N. Hu, Electroanalysis 16 (2004) 1122e1131.[27] M. Senel, M.F. Abasıyanık, Electroanalysis 22 (2010) 1765e1771.

[28] S. Mahiuddin, A. Renoncourt, P. Bauduin, D. Touraud, W. Kunz, Langmuir 21(2005) 5259e5262.

[29] M. Senel, A. Coskun, M.F. Abasıyanık, A. Bozkurt, Chem Pap 64 (2010) 1e7.[30] P.B. Beata, M. Jan, L. Andrzej, Electrochim. Acta 51 (2006) 2173e2181.[31] K. Uzun, E. Çevik, M. Senel, H. Sözeri, A. Baykal, M.F. Abasıyanık, M.S. Toprak,

J. Nanoparticle Res. 12 (2010) 3057e3067.[32] S. Uppuluri, D.R. Swanson, L.T. Piehler, J. Li, G.L. Hagnauer, D.A. Tomalia, Adv.

Mater. 12 (2000) 796e800.[33] M. Senel, M. Tülü, A. Bozkurt, Cent Euro J Chem 5 (2007) 546e556.[34] M. Tülü, M. Senel, J. Appl. Polym. Sci. 109 (2008) 2808e2814.[35] E. Bustos, J. Manriques, E. Juarista, T.W. Chapman, L.A. Godinez, J. Braz. Chem.

Soc. 19 (2008) 1010e1016.[36] H.L. Zhang, G.S. Lai, D.Y. Han, A.M. Yu, Anal. Bioanal. Chem. 390 (2008)

971e977.[37] M. Senel, E. Çevik, M.F. Abasıyanık, Sens. Actuators, B 145 (2010) 444e450.[38] F.H. Wu, Z.H. Hua, J.J. Xua, Y. Tian, L.W. Wang, Y.Z. Xian, L.T. Jin, Electrochim.

Acta 53 (2008) 8238e8244.[39] L.H. Liua, F.Q. Zhao, L.Q. Liua, J. Lia, B.Z. Zeng, Colloids Surf., B 68 (2009) 93e97.[40] R.A. Kamin, G.S. Wilson, Anal. Chem. 52 (1980) 1198e1205.[41] A. Mulchandani, S. Pan, Anal. Biochem. 267 (1999) 141e147.[42] G. H Wang, L.M. Zhang, J. Phys. Chem. B 110 (2006) 24684e24868.[43] A. Garcia, C. Peniche-Covas, B. Chico, B.K. Simpson, R. Villalonga, Macromol.

Biosci. 7 (2007) 435e439.[44] A.K.M. Kafi, G. Wu, A. Chen, Biosens. Bioelectron. 24 (2008) 566e571.[45] F.S. Yardımcı, M. Senel, A. Baykal, Mater. Sci. Eng., C 32 (2012) 269e275.