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New Amperometric Cholesterol Biosensors UsingPoly(ethyleneoxide) Conducting PolymersHuseyin Bekir Yildiz a , Dilek Odaci Demirkol b , Serkan Sayin c , Mustafa Yilmaz c , OzcanKoysuren d & Musa Kamaci aa Department of Chemistry , Karamanoglu Mehmetbey University , Karaman , Turkeyb Ege University, Faculty of Science , Biochemistry Department , Bornova , Turkeyc Department of Chemistry , Selcuk University , Konya , Turkeyd Department of Chemical Engineering , Selcuk University , Konya , TurkeyPublished online: 10 Sep 2013.

To cite this article: Huseyin Bekir Yildiz , Dilek Odaci Demirkol , Serkan Sayin , Mustafa Yilmaz , Ozcan Koysuren & MusaKamaci (2013) New Amperometric Cholesterol Biosensors Using Poly(ethyleneoxide) Conducting Polymers, Journal ofMacromolecular Science, Part A: Pure and Applied Chemistry, 50:10, 1075-1084, DOI: 10.1080/10601325.2013.821921

To link to this article: http://dx.doi.org/10.1080/10601325.2013.821921

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Journal of Macromolecular Science, Part A: Pure and Applied Chemistry (2013) 50, 1075–1084Copyright C© Taylor & Francis Group, LLCISSN: 1060-1325 print / 1520-5738 onlineDOI: 10.1080/10601325.2013.821921

New Amperometric Cholesterol Biosensors UsingPoly(ethyleneoxide) Conducting Polymers

HUSEYIN BEKIR YILDIZ1∗, DILEK ODACI DEMIRKOL2, SERKAN SAYIN3, MUSTAFA YILMAZ3,OZCAN KOYSUREN4, and MUSA KAMACI1

1Department of Chemistry, Karamanoglu Mehmetbey University, Karaman, Turkey2Ege University, Faculty of Science, Biochemistry Department, Bornova, Turkey3Department of Chemistry, Selcuk University, Konya, Turkey4Department of Chemical Engineering, Selcuk University, Konya, Turkey

Received March 2013, Accepted May 2013

Accumulation of cholesterol in human blood can cause several health problems such as heart disease, coronary artery disease,arteriosclerosis, hypertension, cerebral thrombosis, etc. Therefore, simple and fast cholesterol determination in blood is clinicallyimportant. In this study, two types of amperometric cholesterol biosensors were designed by physically entrapping cholesterol oxidasein conducting polymers; thiophene capped poly(ethyleneoxide)/polypyrrole (PEO-co-PPy) and 3-methylthienyl methacrylate-co-p-vinyl benzyloxy poly(ethyleneoxide)/polypyrrole (CP-co-PPy). PEO-co-PPy and CP-co-PPy were synthesized electrochemicallyand cholesterol oxidase was immobilized by entrapment during electropolymerization. The amperometric responses of the enzymeelectrodes were measured by monitoring oxidation current of H2O2 at +0.7 V in the absence of a mediator. Kinetic parameters, suchas Km and Imax, operational and storage stabilities, effects of pH and temperature were determined for both entrapment supports.Km values were found as 1.47 and 5.16 mM for PEO-co-PPy and CP-co-PPy enzyme electrodes, respectively. By using these Kmvalues, it can be observed that ChOx immobilized in PEO-co-PPy shows higher affinity towards the substrate.

Keywords: Amperometric biosensors, cholesterol sensors, cholesterol oxidase, conducting polymers, enzyme immobilization

1 Introduction

A biosensor is a chemical sensor comprising from abioreceptor part and a transducer part (1). Bioreceptor ofthe biosensor is a biological molecular species or a livingorganism which is immobilized on a solid surface andutilizes a biochemical mechanism for recognition (2). Themain advantage of a biosensor is its selectivity towardsan analyte which is specific chemical or chemicals (3) ina complex sample. The signals recorded depend on theconcentration of the analyte and they can be interpretedin terms of changes in resonance unit (4, 5), UV-Vis-IRabsorption (6, 7), mass (8, 9), electrical (10, 11) andphotoelectrical properties (12, 13). Enzyme biosensorshave become increasingly important in clinical diagnostics,food industries and environmental control (14). Enzymesare expensive. Therefore, it is difficult and costly to separatethem from the reaction mixture. However, the advantages

∗Address correspondence to: Huseyin Bekir Yildiz, Departmentof Chemistry, Karamanoglu Mehmetbey University, 70100 Kara-man, Turkey. Tel: +90 338 226 20 00; Fax: +90 338 226 21 16;E-mail: yildizhb@kmu.edu.tr

of immobilized are repeated use, easy separation from theproduct environment, enhanced stability and reduction inthe cost of operation (15).

The amount of cholesterol in more than normal val-ues which are in the range of 1.3–2.6 mg/mL in humanblood can cause various clinical disorders, such as heartdisease, coronary artery disease, arteriosclerosis, hyperten-sion, cerebral thrombosis, etc. (16). Moreover, cholesterol isalso known to play an important role in the brain synapsesand in the immune system. Therefore, the determination ofcholesterol levels is of great importance in clinical analy-sis/diagnosis (17). Cholesterol oxidase is the enzyme whichis mainly preferred and used in the cholesterol determina-tion studies. Cholesterol oxidase (E.C. 1.1.3.6) is a flavinadenosine dinucleotide (FAD)-containing enzyme and itcatalyzes the oxidation of cholesterol into cholest-4-en-3-one and hydrogen peroxide (Sch 1). Many analytical meth-ods have been developed during the years for the deter-mination of cholesterol. These include the use of chemicalmethods such as, colorimetric methods, chromatographicand spectroscopic methods. Although, some of these meth-ods are precise and reliable, they are complex, time con-suming and require previous separation processes, expen-sive instrumentation and trained operators (18, 19). The

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1076 Yildiz et al.

CC

CO

CH2

S

CH3

CH2

O

N

H

SN

NNN

H HHH

n

CH3OCH2CH2(OCH2CH2)nCH2

H

m

CC

CO

CH2

CH3

CH2

O

CH3OCH2CH2(OCH2CH2)nCH2

H

m

Sch. 1. Structure of CP-co-PPy conducting copolymer (6, 29, 36,46).

electrochemical technique which is another well-knownbiochemical detection method for cholesterol provides fast,simple, low cost and high performance detection (20).Chronoamperometry is the much desired technique in elec-trochemical detection systems for cholesterol, as either theoxygen consumption or the hydrogen peroxide productioncan be monitored by using this technique.

After the first discovery of the conducting polymers, anew period has been started for the material and macro-molecular sciences. Conducting polymers have been used inmany applications such as such as electrochromic materials(21) organic-based solar cells (22, 23), organic field effecttransistors (24, 25), organic light-emitting diodes (26), drugrelease systems (27), rechargeable batteries (28) and immo-bilization of biomolecules (29). Conducting polymers arehighly desirable in biosensor design because of their com-patibility with biological molecules, easy preparation, highreproducibility and electrochemical properties (30–32). Be-sides, they can have very well-organized molecular structureand therefore they can act as a three dimensional matrixfor biomolecule immobilization (33–35). Immobilizationof a biomolecule in electropolymerized films is a simpleone-step method. In this physical entrapment, an appropri-ate potential is applied to the working electrode immersedin aqueous solution containing the biomolecule and theelectropolymerizable monomer. This method enables re-

S

CH2CH2 OH

N

H

SN

NNN

O

H HHH

H

n

CH2CH2

n H

n

Sch. 2. Structure of PEO-co-PPy conducting copolymer (29, 37,46).

producible and precise formation of polymeric films withcontrolled thickness and morphology.

The present work describes two amperometric choles-terol biosensors which can determine cholesterol sim-ple and fast by using poly(ethyleneoxide) conductingpolymers. Cholesterol oxidase enzyme was entrappedin thiophene capped poly(ethyleneoxide)/polypyrrole(PEO-co-PPy) and 3-methylthienyl methacrylate-co-p-vinylbenzyloxy poly(ethyleneoxide)/polypyrrole (CP-co-PPy) matrices (Schs 1, 2 and 3). CP and PEO and theirconducting copolymers of pyrrole and thiophene were syn-thesized and characterized in the previous studies (36, 37).The biosensor responses were registered as the current sig-nals (μA) via measuring oxidation current of H2O2 at+0.7 V vs Ag/AgCl (4 M KCl) in the presence of choles-terol substrate without using a mediator (Sch 4). Althoughmediators enhance the sensitivity and selectivity (38–40),cholesterol sensors produced have enough sensitivity andselectivity towards the substrate; hence, a mediator was notused in this study. The biosensors were characterized interms of several parameters such as operational and stor-age stabilities, kinetic parameters (Km and Imax), effectsof pH and temperature.

2 Experimental

2.1 Material and Reagents

Cholesterol oxidase [E.C.1.1.3.6] (26.4 U/mg protein) fromPseudomonas fluorescens, cholesterol, sodium dodecyl sul-fate (SDS) and Triton X-100 were purchased from SigmaAldrich and used with no further purification. Pyrrole

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New Cholesterol Biosensors Via Conducting Polymers 1077

Sch. 3. Schematic representation of immobilization of cholesterol oxidase in conducting copolymer matrices. (Color figure availableonline.)

and hydrogen peroxide were supplied by Merck. Ethanol(Merck) was of analytical grade and used as received forpreparing the cholesterol stock solution (0.05 M). Phos-phate buffer (pH = 7) for the electrosynthesis was preparedby dissolving 0.025 mol of Na2HPO4 (Fisher ScientificCompany) and NaH2PO4 (Fischer Scientific Company) in

Sch. 4. The mechanism of amperometric cholesterol detection.

1 L distilled water. On the other hand, the phosphate buffer(pH = 7) utilized in the amperometric measurements con-sisted of 0.04 M Na2HPO4, 0.04 M KH2PO4 (Merck), and0.1 M KCl (Fisher Scientific Company) to provide ionicconductivity.

2.2 Instrumentation

Electrochemical measurements were carried out at ambientconditions (∼25◦C) in a cell equipped with Ag/AgCl ref-erence electrode (silver wire dipped in 4 M KCl saturatedwith silver chloride, Fischer Scientific Company), platinum(Pt) plate working and counter electrodes with 0.12-cm2

area each. All the electrosynthesis and amperometric ex-periments were carried out with Ivium Compact Stat (TheNetherlands) potentiostat via chronoamperometry. JEOLJSM-6400 model scanning electron microscope (SEM) wasalso used for the characterization of the biosensors.

2.3 Preparation of PEO-co-PPy/ChOx andCP-co-PPy/ChOx Enzyme Electrodes

Cholesterol oxidase (ChOx) was immobilized in two dif-ferent conducting polymer matrices via constant poten-tial application. Preparation of CP-co-PPy/ChOx enzyme

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1078 Yildiz et al.

electrode was performed by applying +1.0 V at room tem-perature in a typical three-electrode cell containing CPcoated platinum foil (1 cm2) as working, bare Pt as counterand Ag/Ag+ as reference electrodes. Electrolysis solu-tion for ChOx immobilization consists of ChOx (0.8 mg/10 mL), SDS (1.2 mg/mL), pyrrole (0.01 M) and phosphatebuffer (10 mL, pH 7.0). For the immobilization of ChOxin PEO-co-PPy matrix, a solution of 1.0 mg/10 mL ChOx,2 mg/mL PEO, 1.2 mg/mL supporting electrolyte (SDS),0.01 M pyrrole and 10 mL phosphate buffer (pH 7.0) wasput in a typical three electrode cell. The three electrodecell has the bare Pt as working and counter electrodes anda Ag/Ag+ (4 M) reference electrode. Polymerization reac-tions for preparing of PEO-co-PPy/ChOx electrode werecarried out by applying 1.0V. SDS is not only the support-ing electrolyte for the electrosynthesis, but it also enhancessolubility of pyrrole (Py) in aqueous solutions, thanks toits ionic surfactant property. After electrolysis, the enzymeelectrodes were washed with distilled water in order to re-move both excess supporting electrolyte and unbound en-zyme and kept in phosphate buffer (pH 7.0) at 4◦C whennot in use.

2.4 Preparation of Cholesterol Solution

Cholesterol stock solution (0.05 M) was prepared dissolv-ing 0.387 g of cholesterol in 20 ml of ethanol at room tem-perature via gently mixing with constant speed to obtain aclear solution. The stock solution was stored at +4◦C in thedark and consumed in 10 days. Triton X-100, the nonionicsurfactant providing solubility of cholesterol in aqueoussolutions, was added to the cholesterol solutions just be-fore the experiments. High Triton X-100 concentration caninhibit the activity of ChOx. The concentration range of0.8–1.2% (v/v) was found to be suitable by Tan et al. (41).For all the experiments, this surfactant was added in theratio of 1% (v/v) to the analyte solutions.

2.5 Measurements

All experiments were carried out at ambient conditionsin an electrochemical cell containing 10 mL of phosphatebuffer as previously described section of Material andreagents. After each run, the electrode was washed withdistilled water. The biosensor was initially equilibrated inbuffer and then the substrate was added to the medium.Current response due to cholesterol addition was recordedat the 150th second. The amperometric responses were mea-sured via monitoring oxidation current of H2O2 at +0.7 Vwithout using a mediator. Initially, the baseline currentbecame constant, and then the analyte was added to themedium; the current immediately increased (response timeswere 2–3 s) and reached a steady state almost at the endof 150–200 s. Finally, the differences between these currentvalues were recorded. During the experiments, the systemwas gently stirred.

2.6 Determination of Optimum pH and OptimumTemperature

The reaction temperature was changed between 10◦C and60◦C while the cholesterol concentration was kept constantat 10 Km for every case. For pH optimization at 25◦C, (inorder to be able to use these electrodes as biosensors) the pHof the reaction was altered between pH 4 and pH 11 whilethe cholesterol concentration was kept constant at 10 Km.In all experiments for both pH and temperature optimiza-tions, the enzyme activity determination experiments wereperformed via application of +0.7 V as previously describedin the Measurements section. Relative enzyme activity wascalculated by assigning the maximum value of activity as100% in determination of optimum pH experiments.

2.7 Operational and Storage Stability Experiments

The operational stability of electrodes was studied by per-forming 20 repetitive measurements in the same day. Stor-age stability of enzyme electrodes was determined by check-ing the activities every day for a week and then once in5 days throughout 30 days. In the investigations of bothoperational and storage stabilities, enzyme activity deter-mination was found via application of +0.7 V at pH 7.0and 25◦C as previously described in the Measurements sec-tion. Substrate concentrations were kept at 10 Km andelectrodes were stored in buffer solution at 4◦C when notin use. As done in determination of optimum pH experi-ments, relative enzyme activity was calculated by assigningthe maximum values of activity as 100% in operational andstorage stability experiments.

3 Results and Discussion

3.1 Optimization of Polymer Film Thickness

The effect of film thickness on following the oxygen con-sumption was initially studied. Deciding on the optimalthickness of the polymer film for making biosensors is veryimportant. There are two reasons why this is important.One of them is that very thin polymeric films may be unableto protect the enzyme from the environmental effects. Theother one, however, very thick films may complicate the dif-fusion process between solution and entrapment support,and as a result, the substrate may not associate with therecognition element. The thickness was controlled by fixingthe charge at which the maximum amperometric responsewas obtained. First, CP coated Pt electrodes were elec-tropolymerized with PPy depositing 1Q (0.0428 C-chargedeposited in a minute), 2Q (0.0856 C), 3Q (0.128 C), and4Q (0.171 C) and then the experiments were carried out byadding 5 mL H2O2 (0.222mM in a total volume of 15 ml)to medium. The current responses were found as 19, 32,43, and 27μA/cm2 for 1Q, 2Q, 3Q and 4Q CP-co-PPy elec-trodes, respectively. In the case of PEO-co-PPy,after 1Q

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New Cholesterol Biosensors Via Conducting Polymers 1079

(0.0428 C), 2Q (0.0856 C), 3Q (0.128 C), 4Q (0.171 C)and 5Q (0.214 C) deposition, the experiments were doneby adding 5 mL hydrogen peroxide (0.222 mM in a totalvolume of 15 mL). The current responses were obtainedas 21, 33, 45, 57 and 51 μA/cm2 for 1Q, 2Q, 3Q, 4Q and5Q PEO-co-PPy electrodes, respectively. In summary, inall of the subsequent experiments, 3Q CP-co-PPy (nearly80 μm thick) and 4Q PEO-co-PPy (nearly 110 μm thick)electrodes were utilized. The thickness of the polymers wasestimated using the charge required for the film coatingon the electrode surfaces. These values were checked witha micrometer after peeling off the polymer film from theelectrode surface.

3.2 Enzyme Loading

Performance of the biosensor strongly depends on theamount of the immobilized enzymes since this would af-fect biosensor activity directly. To determine the optimalamount of enzyme loading, different biosensors contain-ing 0.4, 0.6, 0.8, 1.0 and 1.2 mg ChOx were prepared andthe responses of the enzyme electrodes containing differ-ent amounts of ChOx in the presence of 10 mM choles-terol were studied. The current response rose with increas-ing enzyme loading and then reached a saturation point.The maximum responses were obtained with 0.8 mg pro-tein/10 ml and 1.0 mg protein/10 ml for CP-co-PPy andPEO-co-PPy enzyme electrodes, respectively. In conclu-sion, in the preparation of 3Q CP-co-PPy enzyme elec-trodes, 0.8 mg protein/10 ml was added to the polymeriza-tion medium,whereas 4Q PEO-co-PPy was loaded 1.0 mgprotein/10 ml to obtain the highest sensitivity. The resultsfor both sensors are shown in Figure 1.

3.3 Kinetic Parameters

Kinetic studies of the immobilized ChOx were performed atat constant temperature (25◦C) and pH (pH 7) while vary-

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.40

5

10

15

20

25

30

35

40

45

Cu

rren

t R

esp

on

se (µ

A/c

m2 )

Protein Concentration (mg/10 mL)

Fig. 1. Current responses containing different amounts of ChOxin the presence of 10 mM cholesterol solution for CP-co-PPy/ChOx (�) and PEO-co-PPy/ChOx (◦) electrodes. (Colorfigure available online.)

ing cholesterol concentration. In the equilibrium modelof Michaelis–Menten, the substrate binding step is as-sumed to be fast relative to the rate of breakdown of theenzyme–substrate complex (30). Kinetic parameters for theamperometric biosensors include the maximum reactionrate (Imax) of the enzymatic reaction and the Michaelis-Menten constant (Km) which is the equilibrium dissoci-ation constant for the complex. The Michaelis-Mentenconstant (Km), defines the affinity of enzyme toward itssubstrate corresponds to substrate concentration at 1/2Imax (42). The lower the Km value means the higher itsaffinity against its substrate. The kinetic parameters, Imaxand Km, were obtained from the Lineweaver-Burk plotwhich is a plot of 1/Vo against 1/[Substrate] for systemsobeying the Michaelis-Menten equation. The graph be-ing linear can be extrapolated at anywhere approximat-ing to a saturating substrate concentration, even if noexperiment has been performed and from the extrapo-lated graph, the values of Km and Vmax can be deter-mined (6). Increasing current responses with the increas-ing substrate concentration for both PEO-co-PPy/ChOxand CP-co-PPy/ChOx electrodes were shown at Figure 2aand Figure 3a, respectively. Figure 2b and Figure 3b givethe Lineweaver–Burk plots of these enzyme electrodes. Thecurrent responses that belong to minimum detectable con-centrations were obtained as 3.5 and 3.9 μA/cm2 for CP-co-PPy/ChOx and PEO-co-PPy/ChOx electrodes respec-tively. Lower the Km value means higher affinity of enzymefor its substrate. Therefore, the reason for the lower Kmvalue of PEO-co-PPy/ChOx electrode than that of CP-co-PPy/ChOx can be explained as that more ChOx wasassociated with cholesterol in PEO-co-PPy matrix. Sensi-tivity of an enzyme electrode can be described as Imax/Kmratio (43, 44) and sensitivities were calculated as 11.87 and13.32 μA/mM.cm2 for CP-co-PPy/ChOx and PEO-co-PPy/ChOx enzyme electrodes, respectively (Table 1). It canbe assumed that with small Km value and comparativelyhigh sensitivity, the PEO-co-PPy/ChOx enzyme electrodeshowed better biosensor characteristics.

3.4 Effect of pH

The pH stabilities of the freshly prepared enzyme electrodeswere determined via application of +0.7 V at different pHsranging from 4 to 11 at 25◦C. Due to the denaturationof ChOx in acidic media lower than pH 4, the pH opti-mization study started from pH 4 for enzyme electrodes.

Table 1. Kinetic parameters of immobilized cholesterol oxidase

Imax Sensitivity(μA/cm2) Km (mM) (μA/mM.cm2)

PEO-co-PPy/ChOx 19.58 1.47 13.32CP-co-PPy/ChOx 61.26 5.16 11.87

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0.0 0.5 1.0 1.5 2.0 2.5 3.00.00

0.05

0.10

0.15

0.20

0.25

0.30

(b)

1/C

urr

ent

Res

po

nse

1/Concentration

y= 0.047 + 0.085x

R2 = 0.994

0 2 4 6 8 10 12 140

2

4

6

8

10

12

14

16

18

20

(a)

Cu

rren

t R

esp

on

se (

µA/c

m2 )

Concentration (mM)

Fig. 2. (a) Current response vs concentration and, (b) 1/Currentresponse vs. 1/concentration for the PEO-co-PPy/ChOx enzymeelectrode (at pH 7 and 25◦C). (Color figure available online.)

0 2 4 6 8 10 12 140

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50

(a)

Cu

rren

t R

esp

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se (

µA/c

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Concentration (mM)

0.0 0.5 1.0 1.5 2.0 2.5 3.00.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

(b)

y= 0.005 + 0.112x

R2 = 0.991

1/C

urr

ent

Res

po

nse

1/Concentration

Fig. 3. (a) Current response vs concentration and, (b) 1/Currentresponse vs. 1/concentration for the CP-co-PPy/ChOx enzymeelectrode (at pH 7 and 25◦C). (Color figure available online.)

0 2 4 6 8 10 120

20

40

60

80

100

120

Rel

ativ

e E

nzy

me

Act

ivit

y

pH

Fig. 4. Effect of pH on activity of cholesterol oxidase immobilizedin CP-co-PPy (�) and PEO-co-PPy (◦) matrices. (Color figureavailable online.)

The maximum activities were observed at pH 6.0 and 7.0for CP-co-PPy and PEO-co-PPy matrices respectively. Theyare illustrated in Figure 4. Up to pH 11, although CP-co-PPy matrix lost 66% of its activity PEO-co-PPy matrix lostonly 21% of its activity. Moreover, PEO-co-PPy matrix wasstable on the pH range between the pH 6 and 10 and hence itcan be used reliably at high pH values for enzyme reactions.Moreover, due to the high stability of PEO-co-PPy/ChOxenzyme electrode on the pH range between pH 6 and 10,it has also the advantage in medical applications since theblood pH is 7.4. The optimum pHs were shifted towardsthe alkaline side when compared with the soluble enzyme.This might be explained by partitioning of protons. Nega-tively charged groups of the matrix will tend to concentrateprotons, and this causes lowering the pH around the en-zyme. Therefore, the pH around the enzyme will be lowerthan that of the bulk phase from which the measurementof pH is carried out.

3.5 Effect of Temperature

The enzyme activity strongly depends on temperature be-cause very hot or very cold conditions can inactivate the en-zyme. The temperature stabilities of the freshly prepared en-zyme electrodes were determined via application of +0.7 Vat different temperatures ranging from 10 to 60◦C in phos-phate buffer (pH 7.0). Because of using ethanol for dis-solving cholesterol, activities were not checked at highertemperatures to prevent vaporization. As illustrated inFigure 5a and Figure 6a, current response gradually in-creased with increasing temperature and reached a maxi-mum at 50◦C. It can be understood from Figure 6a that CP-co-PPy/ChOx electrode showed high intensity responsesas predicted due to its high Imax. These results demon-strated that both enzyme electrodes showed the same trend.By using the Arrhenius equation, which is I(T) = I0 exp(Ea / RT) and plotting Ln current response vs. 1/T graphs,the activation energies for the enzymatic reactions in CP-co-PPy and PEO-co-PPy matrices were calculated as 16.8and 12.9 kJ/mol, respectively (Fig. 5b and 6b). The smaller

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New Cholesterol Biosensors Via Conducting Polymers 1081

0 10 20 30 40 50 60 700

4

8

12

16

20

(a)C

urr

ent

Res

po

nse

(µA

/cm

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Temperature (°C)

0.0030 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036

2.0

2.2

2.4

2.6

2.8

3.0

(b)

y = 7.60 - 1548.88 X

Ln

(cu

rren

t re

spo

nse

, µA

/cm

2 )

1/Temperature (K-1)

Fig. 5. (a) Effect incubation temperature on activity of choles-terol oxidase immobilized in PEO-co-PPy matrix and, (b) Deter-mination of the activation energy for the enzymatic reaction inPEO-co-PPy. (Color figure available online.)

Ea means that the ChOx enzyme entrapped in PEO-co-PPymatrix shows higher enzyme activity and the sensor exhibitshigher affinity towards its substrate, which is in agreementwith lower Km value.

3.6 Operational and Storage Stabilities of EnzymeElectrodes

Enzymes can easily lose their catalytic activity and dena-tured. Biomolecules like enzymes have limited stabilitiesespecially when they are removed from their native areasand their stabilities and performances decrease due to im-mobilization. Therefore, operational and storage stabilityare important considerations for an immobilized enzyme.Operational and storage stabilities were shown in Figures 7and 8, respectively. Operational stability of enzyme elec-trodes was tried to estimate the stability of electrodes interms of 20 repetitive uses. PEO-co-PPy/ChOx electrodemaintained an activity at 80% until the assay number 7 andexhibited a good stability upon the repetitive uses. After6th assay, enzyme activity decreased and after 16th assay

0 10 20 30 40 50 60 700

10

20

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(a)

Temperature (°C)

Cu

rren

t R

esp

on

se ( µ

A/c

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0.0030 0.0031 0.0032 0.0033 0.0034 0.0035 0.00362.4

2.6

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3.0

3.2

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3.8

4.0

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Ln

(cu

rren

t re

spo

nse

, µA

/cm

2 )

1/Temperature (K-1)

y = 9.93 - 2017.59 X

Fig. 6. (a) Effect incubation temperature on activity of cholesteroloxidase immobilized in CP-co-PPy matrix and, (b) Determinationof the activation energy for the enzymatic reaction in CP-co-PPy.(Color figure available online.)

it stayed constant at 50% of its original activity. CP-co-PPy/ChOx enzyme electrode showed very high operationalstability and retained 95% of its original activity until theassay number 9 and then kept 85% of its activity even af-ter 20th use. It was observed that although PEO-co-PPy

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Fig. 7. Operational stabilities of CP-co-PPy/ChOx (�) and PEO-co-PPy/ChOx (◦) electrodes. (Color figure available online.)

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matrix was likely to protect the enzyme better, as it is thickerthan CP-co-PPy matrix, it is more vulnerable to environ-mental effects. Besides, the slight increase in the responseof CP-co-PPy/ChOx electrode is related to the swelling ofthe polymer structure and it was speculated that swelling ofthe polymer may cause changing positions of the enzymemolecules in the polymer to increase the enzyme activityslightly. As known, due to physical entrapment, there is nobond between enzymes and the polymer in the polymer andwhen the polymer swells, the position changing for enzymemolecules can occur (45).

Storage stability of immobilized ChOx in PEO-co-PPyexhibited a 55% loss of its activity in 20 days and stayedconstant until the end of its storage stability experiment. Onthe other hand, immobilized ChOx in CP-co-PPy matrixlost 15% of its activity in the first 15 days and then stayedconstant with 85% of its original activity until the 30th day.In spite of having good enzyme protection against hightemperature and pH, PEO-co-PPy/ChOx enzyme electrodeshowed worse storage stability than the CP-co-PPy/ChOxenzyme electrode. This can be explained that some amountof enzyme dropped to the solution from the matrix whilebeing studied. Both electrodes have very good stabilities inthe first 5 days and can be safely used in this period. Sinceimmobilized ChOx in CP-co-PPy maintained 85% of itsoriginal activity after the 15th day and stayed constant upto the 30th day, it can also be used safely between the 15th

and 30th days with a very high activity.

3.7 Morphologies of Films

In order to determine the surface morphologies of polymerfilms, scanning electron microscopy (SEM) technique wasused. Scanning electron micrographs of CP-co-PPy/ChOxand PEO-co-PPy/ChOx electrodes are given in Figure 9.The surface properties of CP-co-PPy and PEO-co-PPy ma-trices without yeast cells were given in previous studies(36, 37). The surface morphologies of these films werecompletely different compared to the films prepared inthe absence of yeast cells. Cauliflower-like structure was

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Fig. 8. Storage stability of CP-co-PPy/ChOx (�) and PEO-co-PPy/ChOx (◦) electrodes. (Color figure available online.)

Fig. 9. Scanning electron micrographs of (a) CP-co-PPy matrixwith ChOx and (b) PEO-co-PPy matrix with ChOx.

noticeably damaged when yeast cells were entrapped inthese matrices.

4 Conclusion

The redox enzyme, cholesterol oxidase, which catalyzesthe oxidation of β-D-cholesterol to D-glucono-1,5-lactoneand hydrogen peroxide, was immobilized in two differ-ent poly(ethyleneoxide) type matrices for the first time to

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New Cholesterol Biosensors Via Conducting Polymers 1083

construct amperometric biosensors. The amperometric re-sponses of CP-co-PPy /ChOx and PEO-co-PPy/ChOx en-zyme electrodes were measured by monitoring oxidationcurrent of H2O2 at +0.7 V in the absence of a mediatorKinetic parameters, operational and storage stabilities, op-timum temperature and pH were investigated for the ma-trices. PEO-co-PPy/ChOx electrode had smaller Km andImax values and higher sensitivity when compared with Kmand Vmax values of free enzyme and CP-co-PPy/tyrosinaseelectrode. The smaller Km value shows that PEO-co-PPymatrix provides a microenvironment which is more suit-able than that in the solution and CP-co-PPy matrix. It canbe understood from higher sensivitiy that enzyme immo-bilized in PEO-co-PPy matrix has higher affinity towardsits substrate than the enzyme immobilized in CP-co-PPymatrix does. In spite of having high temperature and pHstabilities, operational and storage stabilities of PEO-co-PPy/ChOx enzyme electrode were not good and the rea-son of the bad stabilities can be interpreted that this ma-trix could not protect enzyme well and some of enzymedropped to solution from the matrix. Moreover, immobi-lization of cholesterol oxidase enzyme in these conductingpolymer electrodes can be studied as an alternative biosen-sor fabrication for the determination of cholesterol amountin fruit juices without requiring sample pre-treatment andresults show that this significant development can success-fully replace the classical methods. This study proves thatconducting polymers; CP-co-PPy and PEO-co-PPy can beused as immobilization matrices for ChOx to produce am-perometric biosensors which can determine the cholesterolamount in the real samples fast and sensitive.

Acknowledgments

Authors would like to thank the Scientific and Technolog-ical Research Council of Turkey (TUBITAK Grant Num-ber 109T439) and the Scientific Research Projects Founda-tion of Karamanoglu Mehmetbey University (KMU-BAPGrant Number 09-M-11) for the financial support of thisresearch.

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