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Sensors and Actuators B 176 (2013) 299– 306

Contents lists available at SciVerse ScienceDirect

Sensors and Actuators B: Chemical

journa l h o mepage: www.elsev ier .com/ locate /snb

ovel reagentless glucose biosensor based on ferrocene cored asymmetricAMAM dendrimers

ehmet S enela,∗, Cevdet Nergiza,∗, Emre C evikb

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, Turkey

r t i c l e i n f o

rticle history:eceived 10 August 2012eceived in revised form 9 October 2012ccepted 13 October 2012

a b s t r a c t

A novel amperometric glucose biosensor is fabricated with immobilization of glucose oxidase (GOx) ontoPAMAM-Fc dendrimers decorated gold electrode. Series of asymmetric PAMAM dendrimers containinga single ferrocene unit located in the focal point of these macromolecules have been synthesized andcharacterized. Surface of the gold electrode was covalently modified with 3-mercaptopropionic acid,

vailable online xxx

eywords:lucose oxidaseiosensorAMAM dendrimer

PAMAM-Fc dendrimers and GOx enzyme by using coupling agents, respectively. The PAMAM-Fc/GOxbiosensor shows an excellent performance for glucose at +0.25 V with a high sensitivity (6.54 �A/mM)and lower response time (∼3 s) in a wide concentration range of 1–22 mM (correlation coefficient of0.9988). In addition, PAMAM-Fc/GOx biosensor possesses better reproducibility, storage stability andthere is negligible interference from other electroactive components.

errocene

. Introduction

In the field of amperometric oxidoreductase electrodes, muchffort is currently being aimed at the immobilization of redoxediators that shuttle electrons between the enzyme’s redox

enters and the electrode (or transducer) [1,2]. In recent years, lot of studies have been done for the construction of elec-ron transfer interface between active site of the enzymend electrode surface to apply them high-performance amper-metric enzyme based biosensors [3–7]. To date, ferroceneas been one of the most successful redox mediators due to

ts well-behaved electrochemical properties. For this reason, variety of immobilization procedures for ferrocene speciesave been developed to fabricate reagentless amperometriciosensors [7–9].

Redox proteins often have their prosthetic groups asymmet-ically located and partially buried in the protein’s polypeptideackbone. In electrochemical experiments, the interfacial orien-ation of the protein near the electrode surface influences the

ffective rates of the heterogeneous electron transfer reaction10,11]. Dendrimers are considered as modern regular tree-ike macromolecules. They have controllable microenvironment

∗ Corresponding authors at: Fatih University, Department of Chemistry,uyukcekmece Kampusu, 34500 B.Cekmece/Istanbul, Turkey.el.: +90 2128663300; fax: +90 2128663402.

E-mail addresses: msenel@fatih.edu.tr (M. S enel),nergiz@fatih.edu.tr (C. Nergiz).

925-4005/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.snb.2012.10.072

© 2012 Elsevier B.V. All rights reserved.

around the dendritic core and cooperativity effects between sur-face groups. A lot of reports have been published on dendrimersfunctionalization with peripheral or core [12]. Recently, manyefforts have focused on the redox-active-dendrimers, which maybe measured up to redox proteins [13,14]. The clear advantageof the redox-active-dendrimers; that is, dendrimers are robustmolecules that do not denature, unlike proteins. Shinohara et al.was designed artificial redox proteins by functionalization ofbovine serum albumin (BSA) with ferrocene groups in order todiscuss its applicability for facile electron transferring interfacesbetween redox enzyme and electrode surface. They were demon-strated that the redox group-introduced BSA functioned as redoxproteins [15].

Detection of glucose concentration is a crucial indicator ofmany diseases, such as diabetes, endocrine metabolic disorder.In recent years, many efforts have been made to develop reliableglucose biosensors using electrochemical [16], chemiluminescenceor other methods [17]. Among all the methods, enzyme-involvedelectrochemical glucose biosensor has been intensively studiedbecause of its simplicity, high selectivity and relative low cost[18,19]. In this technique, the enzyme immobilization is consideredto be one of the most important issues. Since the performance of abiosensor [20] much relies on the supporting materials, searchingof materials that provide good environment for the efficientenzyme loading and maintenance of enzyme bioactivity is highly

desired. Currently, the glucose oxidase (GOx) is widely employedin most of the glucose biosensors due to its stability and high selec-tivity to glucose, especially the amperometric glucose biosensors[21–24]. It contains two flavin adenine dinucleotide (FAD) cofactors

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00 M. S enel et al. / Sensors and

nd catalyze the oxidation of glucose according to the followingeaction [25]:

ˇ-D-Glucose + GOx (FAD) → GOx (FADH2) + D-glucono-ı-lactone

GOx (FADH2) + O2 → GOx (FAD) + H2O2

In previous study, PAMAM dendrimer modified with redoxediator, ferrocene, and used for the construction of reagentless

mperometric glucose biosensor [26]. In those studies ferroceneovalently bonded on peripheral amine groups and attached ontolectrode surface. Due to this attachment point of ferrocene, bind-ng affinity of primary amines on dendrimer surface decrease.rimary amines are binding site of the PAMAM dendrimer tohe enzyme molecules. To solve this deficiency problem, noveledox active PAMAM dendrimers were synthesized by startingrom ferrocene unit as a focal point and used for the construc-ion of amperometric biosensor. The effects of generation ofhe ferrocene dendrimers with and without GOx were inves-igated, and the pH-activity profiles of immobilized GOx werexamined. The storage and operational stabilities of the sensorsere established, and the influence of the interferent’s ascor-

ic acid, acetaminophen, and uric acid, which are present inody fluids, on the amperometric signals of the sensors wasetermined.

. Experimental

.1. Materials

Glucose Oxidase (GOx) (EC 1.1.3.4) and 1-ethyl-3-(3-

imethylaminopropyl) carbodiimide hydrochloride (EDC) werebtained from Sigma Chemical Co. Ferrocene carboxyalde-yde was obtained from Aldrich. Glucose, methylacrylate andthylenediamine were obtained from Merck. All other chem-cals were of analytical grade and were used without furtherurification.

Fig. 1. Synthetic pathway of Fc-PAMAM dendrimers (i) ethylenediamine and NaBH4

ors B 176 (2013) 299– 306

2.2. Synthesis of asymmetric ferrocene cored PAMAM dendrimers

A solution of ethylenediamine and formly ferrocene in toluenewas heated to reflux for 6 h. After removing the solvent in vacuo theresidue was solved in ethanol. NaBH4 was added to the mixturewhich was heated reflux for 4 h to reduce double bond betweenC N [27]. The solvent was removed in vacuo and the residue wassuspended in CH2Cl2. The organic layer was washed three timeswith water and dried over Na2SO4.

The amine-terminated ferrocene cored PAMAM dendrimers wassynthesized divergently by initial Michael addition of methano-lic solution of ferrocene amine with excess methyl acrylate (1:10molar ratio) (Fig. 1) [28]. The reaction mixture was stirred forthree days at room temperature. The excess methylacrylate wasremoved under vacuum at 40–50 ◦C temperature to afford theester-functionalized derivative G0.5. The reaction mixture was nextsubmitted to the reaction sequence leading to the next generationFc-PAMAM dendrimer G1.5, consisting of the exhaustive amidationof the ester functionalized G0.5 to ethylenediamine (1:30 molarratio), followed by Michael addition of the resulting amine withmethylacrylate (20 equiv. of G0.5). Excess reagents were removedunder vacuum at 60–70 ◦C temperature. Repetition of this two-step procedure ultimately leads to the next generation Fc-PAMAMdendrimer G3.

2.3. Preparation of GOx immobilized electrodes

Before each new measurements, the gold electrode was polishedwith 1.0, 0.5, and 0.3 �m alumina slurry, sonicated consequen-tially in distilled water and absolute ethanol for 15 min each andetched finally in 0.5 mol L−1 H2SO4 solution by cyclic-potentialscanning between −0.3 and +1.7 V until a reproducible voltam-metric response was obtained. The cleaned gold electrode wasfirst immersed in 20 mmol L−1 3-mercaptopropionic acid (MPA)

aqueous solution for 2 h. After the electrode was thoroughlyrinsed with water to remove physically adsorbed MPA. Fc-PAMAMdendrimers were linked chemically to the functionalized goldelectrodes by promoting the creation of amide bonds NH(CO)

reduction, (ii) methyl acrylate in methanol and ethylenediamine in methanol.

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M. S enel et al. / Sensors and

etween the COOH ends of the MPA and amine peripheralroups (NH2) of the Fc-PAMAM dendrimers. This was achievedy immersing the thiolated gold electrodes in methanolic solu-ions containing 5 × 10−3 mol L−1 EDC (coupling reagent to createmide bonds) and 2.1 × 10−7 mol L−1 Fc-PAMAM dendrimers ofeneration 1.0 (G1), 2.0 (G2) and 3.0 (G3) for 12 h at room temper-ture. Subsequently, the dendrimer-functionalized gold surfacesere washed by dipping them in gently stirred MeOH at room

emperature.Finally, the GOx enzyme was immobilized on the modified elec-

rode by immersing the dendrimer modified gold electrodes inhosphate buffer (pH 7.5) solution that containing 10 mg mL−1

Ox, 5 × 10−3 mol L−1 EDC (coupling reagent to create amideonds) and 21 × 10−6 mol L−1 Fc-PAMAM dendrimers of Genera-ion 1.0 (G1), 2.0 (G2) and 3.0 (G3) for 12 h at +4 ◦C. The resultinglectrode was washed with PBS (pH 7.5) and stored in the same PBSt 4 ◦C when not in use.

.4. Apparatus

The IR absorption spectra (4000–400 cm−1) were recordedith a Mattson Genesis II spectrophotometer. NMR spec-

ra were recorded in CDCl3 using a Bruker 400 MHzpectrometer.

Electrochemical measurements were performed using a CHIodel 842B electrochemical analyzer. A gold plate working elec-

rode (1 cm2), a platinum plate counter electrode (1 cm2), ang/AgCl-saturated KCl reference electrode and a conventional

hree-electrode electrochemical cell were purchased from CHnstruments.

All amperometric measurements were carried out at room tem-

erature in stirred solutions by applying the desired potential andllowing the steady state current to be reached. Once prepared, theOx electrodes were immersed in 10 ml pH 7.5 10 mM PBS solutionnd the amperometric response to the addition of a known amount

Fig. 2. Schematization of the surface of

ors B 176 (2013) 299– 306 301

of glucose solution was recorded. The data shown are the averageof three measurements for each electrode.

3. Results and discussion

3.1. Preparation and characterization of ferrocene-cored PAMAMdendrimers and GOx electrodes

Our strategy for divergent synthesis of ferrocene cored amine-terminated PAMAM dendrimers (G1, G2 and G3) involves the initialMichael reaction ferrocene-amine with methylacrylate followed byexhaustive amidation of the resulting esters with a large excess ofethylenediamine to obtain the next generation with reactive aminegroups. Repetition of this two step procedure ultimately leads todifferent generation PAMAM dendrimers with ferrocene group atthe focal point. The synthesized dendrimers having ferrocene groupat the focal point were used to modify pre-modified (with mer-capto propionic acid) gold electrode by covalent attachment withcoupling agent (Fig. 2). GOx was covalently immobilized onto theartificial redox protein planted gold electrode with coupling agents.

Fig. 3A shows a comparison of the FT-IR spectra between 4000and 400 cm−1 of the Fc-PAMAM dendrimers from G-0 to G-3. Thepeaks around at 3280 cm−1 can be attributed to the stretchingvibration of N-H, indicating the growing of the PAMAM den-drimers on the Fc monomer, other characteristic bands at around1640 cm−1 are assigned to the vibration of the amide group inPAMAM dendrimers. The structure of Fc-PAMAM dendrimer (G3)can be determined from the detailed 1H NMR spectrum (Fig. 3B).In the spectrum, the peaks between 4.08 and 4.11 ppm are specificresonance signals of ferrocene protons. The peaks between 2.2 and3.4 ppm are attributed to the growing of the PAMAM dendrimers

on ferrocene unit, respectively.

The voltammetric behavior of the ferrocene-containing den-drimers between 0.0 and 0.5 V shown in Fig. 4 were obtained atdifferent modified electrodes; (a) Fc-NH2, (b) Fc-PAMAM-G-1, (c)

GOx immobilized gold electrode.

302 M. S enel et al. / Sensors and Actuators B 176 (2013) 299– 306

gener

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Fig. 3. (A) FT-IR spectra of Fc-PAMAM dendrimers with different

c-PAMAM-G-2 and (d) Fc-PAMAM-G-3, respectively. As antici-ated, the electrochemistry of the dendrimers was dominated byhe one-electron, reversible oxidation of the ferrocene nucleus. Ainear correlation between the anodic peak current, Ipa, and squareoot of scan rate, v1/2, was obtained, indicating that charge prop-gation in the dendrons occurs by a diffusion-like process, suchs electron hopping among neighboring redox sites and counter-on motion. During the forward scan, Fe (II) is oxidized to Fe (III),nd subsequently an oxidation current peak is observed. Duringhe reverse scan, Fe (III) is reduced. The difference of the redox

eaks were increased with increasing a scan rate. The voltammet-ic behavior also indicates that ferrocene has been immobilized onhe surface of the glassy carbon electrode [29–31]. The higher theeneration of the dendrimer, the further away the dendritic bulk

able 1he concentration of electroactive ferrocene on modified electrode.

Electrode Eo vs. Ag/AgCl Electroactive Dif

(mV) Fc (mol/cm2) 100

Fc-NH2-G0 240 9.26 × 10−12 39

Fc-PAMAM-G1 238 6.48 × 10−12 33

Fc-PAMAM-G2 220 3.17 × 10−12 22

Fc-PAMAM-G3 226 0.89 × 10−12 16

ations. (B) 1H NMR spectrum of Fc-PAMAM dendritic wedge G3.

keeps the ferrocene subunit from the electrode surface. Overall, ourresults clearly indicate that dendrimer growth extending from oneof the cyclopentadienyl rings of the electroactive ferrocene coretends to hinder kinetically the heterogeneous electron-transferreactions of these dendrimers with the electrode. The concentra-tion of electroactive ferrocene was calculated using Faraday’s lawconsidering the exchanged charge obtained by anodic peak inte-gration, Table 1.

3.2. Optimum pH and temperature

The effect of the pH value of the buffer solution on the responsebehavior of enzyme electrode was studied between pH 5.0 and 9.0and the corresponding results are shown in Fig. 5A. As shown in

ference of the redox peaks (�A) vs. scan rate

200 300 400 500 (mV/s)

70 100 125 15156 75 94 11139 54 70 8632 47 55 70

M. S enel et al. / Sensors and Actuators B 176 (2013) 299– 306 303

F and (d1

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ig. 4. Cyclic voltammograms of (a) Fc-NH2, (b) Fc-PAMAM-G-1, (c) Fc-PAMAM-G-200, 200, 300, 400 and 500 mV/s.

ig. 5A, the response current of the enzyme electrode increasesith increase of pH value and the maximum current response is

bserved at pH 7.5. This is an agreement with that reported initeratures [32]. Therefore, pH 7.5 was chosen for use in furtherxperiments and for the determination of glucose levels.

Also, the effect of the medium temperature was investigatedor another important parameter of enzyme activity in the tem-erature range of 25–55 ◦C, shown in Fig. 5B. The amperometricesponse increases when the temperature rises 25 to 40 ◦C, andhen decreases as the temperature is further increased. The max-mum response appears at about 40 ◦C. At higher temperatures,

he current response decreases slowly due to the denaturation ofhe enzyme. In order to provide real applications conditions, 25 ◦Cas chosen as the operating temperature of the biosensor in the

xperiments.

able 2omparison of the analytical performance of the different GOx electrodes.

Electrode, Aua RTb (s) LRc (mM)

Au/MPA/Fc-NH2-G0 7 1–5

Au/MPA/Fc-PAMAM-G1 5 1–7

Au/MPA/Fc-PAMAM-G2 4 1–10

Au/MPA/Fc-PAMAM-G3 3 1–22

a Au: gold electrode.b RT: response time.c LR: linear range.d DL: detection limit.

) Fc-PAMAM-G-3 electrodes in solution 10 mM PBS solution at different scan rates:

3.3. Electrochemical measurement of glucose

The electrocatalytic behaviors of GOx immobilized electrodewere evaluated by cyclic voltammetry. Fig. 6 shows the cyclicvoltammograms of the modified electrode in pH 7.5 phosphatebuffer saline (PBS) (a) in the absence and (b) presence of glu-cose. In the absence of glucose, only the cyclic voltammogramof Fc dendron was observed. When glucose solution was addedinto the electrochemical cell before measurement, an electrocat-alytic response appeared with an increase of the oxidation current.This result strongly indicate the enzyme-dependent catalytic oxi-

dation of glucose, which originated from the GOx reaction mediatedby a Fc dendron. A new dendritic mediator could effectivelyshuttle electrons from the electrode surface to the redox centerof GOx.

DLd (mM) Sensitivity (�A/mM) KappM (mM)

0.23 5.5 23.510.16 8.7 33.660.33 25.2 22.920.48 32.7 19.86

304 M. S enel et al. / Sensors and Actuators B 176 (2013) 299– 306

Fig. 5. (A) Effect of the pH on the current response of Au/MPA/Fc-PAMAM-G-3/GOxto glucose solution at an applied potential +0.25 V; (B) Effect of temperature on theamperometric response of glucose solution in 10 mM PBS solution, pH 7.5 at anapplied potential of +0.25 V vs. Ag/AgCl.

Fig. 6. Cyclic voltammograms of the Fc-PAMAM-G-3 electrode of (A) in the absenceand (B) presence of glucose at a scan rate of 100 mV/s in pH 7.5 0.01 M PBS solution.

Fig. 7. (A) Amperometric response of Fc-NH2, Fc-PAMAM-G-1, Fc-PAMAM-G-2 andFc-PAMAM-G-3 electrodes to successive addition of 1 mM glucose solution at anapplied potential +0.25 V in stirred 10 mM PBS (pH 7.5; ∼25 ◦C). (B) Calibration

curves for the amperometric response of the biosensing electrodes.

Fig. 7A shows a current–time response plot for the Fc-PAMAMdendrimer (with different generations) based GOx biosensor onsuccessive steps changes of glucose concentration at a workingpotential of 0.25 V. The biosensor exhibited a rapid and sensitiveresponse to the changes in glucose concentration, and the reduc-ing current increased to reach a relatively stable value after ∼3 s. Itcan be seen from Fig. 7, which with increasing the number of gener-ation also amperometric response and sensitivity of the biosensorincreases. The analytical performances of the different modifiedelectrodes are summarized in Table 2. This is also seen from Table 1,when comparison of sensitivity of glucose biosensors based ondendrimers presented. The biosensor based on G0 and G1 den-drimers are characterized with rather low sensitivity. Considerableimprovement of sensitivity takes place when G2 is used instead ofG1. Further increase of sensitivity is obvious for biosensor basedon G3 dendrimer. The increase of the sensitivity of biosensors withincreasing the number of generation of dendrimers can be due tothe increasing of the conductivity and the increasing of the bindingsites for attachment of GOx. Thus, the concentration of GOx shouldbe higher for G3 based biosensor in comparison with that for lower

generation dendrimers. Further, improvement of biosensor sensi-tivity can be achieved by modification of PAMAM dendrimers with

M. S enel et al. / Sensors and Actuators B 176 (2013) 299– 306 305

Fig. 8. (A) Reusability of glucose biosensor (pH 7.5; ∼25 ◦C). (B) Storage stabilityof glucose biosensor. The amperometric responses of these enzyme electrodes areregularly checked during 50 days (pH 7.5; ∼25 ◦C). (C) Amperometric response ofAu∼

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Table 3Glucose content determination in serum samples.

Samples Determined byhospital (mM)

Measured by biosensor(mM)a

RSD (%) Relative error(%)

1 5.51 5.54 ± 0.09 1.7 +0.552 4.85 4.90 ± 0.09 1.8 +1.24

experimental results presented in this work clearly demonstrate

u/MPA/Fc-PAMAM-G-3/GOx electrode to glucose (1.0 mM), ascorbic acid (0.1 mM),ric acid (0.5 mM) and acetaminophen (0.5 mM) in stirred 10 mM PBS (pH 7.5;25 ◦C).

lectron transfer mediator ferrocene as has been reported recently31].

As shown in Fig. 7B and Table 2, under optimum conditions, glu-ose could be determined in a linear range from 1.0 to 22 mM with aorrelation coefficient of 0.9980 and a detection limit of 0.48 mM at

3 5.24 5.16 ± 0.10 1.9 −1.53

a Average of the three measurements.

3 S/N. The apparent Michaelis–Menten constant (KappM ), which gives

an indication of the enzyme–substrate kinetics for the biosensor,was calculated to be 19.86 mM for Au/MPA/Fc-PAMAM-G3, usingthe Lineweaver–Burk equation [33]. Considerably lower Kapp

M valuefor Fc-PAMAM-G3 based biosensor indicates higher affinity of GOxbinding site to the glucose. The all results clearly demonstrate thatthe fast response, high sensitivity and wide linear range can beattributed to the introduction of Fc-PAMAM dendrimers in biosen-sor construction.

3.4. Real sample analysis, reusability, stability and interferencestudies

In order to validate the reliability of the biosensor, the deter-mination of glucose in human serum samples was performedwith the GOx immobilized electrode. Fresh serum samples werefirst analyzed in the local hospital with. The samples were thenre-assayed with the GOx immobilized electrode. For currentmethod, a serum sample was added into the stirred 5 ml PBS (pH7.5), and amperometric response was recorded at +0.25 V. Theresults were shown in Table 3. The glucose levels determinedin this work were close to the values declared by local hospi-tal, indicating that the fabricated glucose biosensor has practicalpotential.

The reusability of successive tests using the same biosensorwas investigated (Fig. 8A). Electrodes were stored at the 4 ◦C(0,1 M; pH 7.5 PBS) for 5 min between each measurement. Theresponse of glucose biosensor gave relatively closed results for firstten measurements, and activity loss 40% was observed with fur-ther application. The stability of Au/MPA/Fc-PAMAM-G-3/GOx wasdetermined by storing the sensor at 4 ◦C for 50 days and monitoringthe response current everyday for glucose at +0.25 V (Fig. 8B). Thebiosensor retained almost its 90% activity after 20 days and then anactivity loss of ∼45% was observed. This good stability behavior wasmost probably due to the stability effect of PAMAM dendrimer’sprotein like structures.

The well known electroactive species in serum were used toevaluate the effect of interring substance on biosensor perfor-mance. Fig. 8C represents the amperometric responses of glucose(Glu) (1 mM) in the presence of ascorbic acid (AA) (1 mM), uric acid(UA) (1 mM) and acetaminophen (AP) (1 mM). The injection of AA,UA and AP did not influence the current response of glucose. Hence,there is negligible interference from other electroactive compo-nents.

4. Conclusions

In this work, we have presented a novel reagentless amper-ometric glucose biosensor based on redox-active-dendrimerswith different generations. Prepared dendrimers were covalentlyattached to the surface of an MPA-modified Au electrode, andGOx was immobilized on this host by covalent bonding. The

that immobilized GOx possesses excellent catalytic ability andwell-retained activity. The results are consistent with efficientenzyme immobilization onto a PAMAM type dendrimer modified

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ences, Fatih University. His current researches are food chemistry, oil technologiesand biosensors.

06 M. S enel et al. / Sensors and

urface containing a ferrocene group linked to a PAMAM den-rimer, which resulted in high enzyme loading, decreased workingotential, increased electrode lifetime and enzyme stability. Theata described in this study demonstrates that Fc-PAMAM den-rimers act as redox active proteins and could work as an electronransferring interface molecules between redox enzyme andlectrode surface. This redox-active-dendrimers might provide aseful platform for incorporation of other enzymes and substrateouples during the construction of biosensors for a variety ofiocatalysis.

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Biographies

Mehmet S enel is an assistant professor in Department of Chemistry, Faculty ofArts and Sciences, Fatih University. He is performing research in the Biotechnol-ogy Research Laboratory on development of biosensors via immobilizing enzymeson polymeric mediators.

Cevdet Nergiz is a professor in Department of Chemistry, Faculty of Arts and Sci-

Emre C evik is now a PhD candidate in Department of Genetics and Bioengineer-ing, Faculty of Engineering, Fatih University. His current researches are enzymeimmobilization and electrochemical biosensors.