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Journal of Macromolecular Science, Part A: Pure and
Applied Chemistry
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New Amperometric Cholesterol Biosensors Using
Poly(ethyleneoxide) Conducting Polymers
Huseyin Bekir Yildiz a , Dilek Odaci Demirkol b , Serkan Sayin c , Mustafa Yilmaz c , Ozcan Koysuren d & Musa Kamaci a
a
Department of Chemistry , Karamanoglu Mehmetbey University , Karaman , Turkey b
Ege University, Faculty of Science , Biochemistry Department , Bornova , Turkey c
Department of Chemistry , Selcuk University , Konya , Turkey d
Department of Chemical Engineering , Selcuk University , Konya , Turkey Published online: 10 Sep 2013.
To cite this article: Huseyin Bekir Yildiz , Dilek Odaci Demirkol , Serkan Sayin , Mustafa Yilmaz , Ozcan Koysuren & Musa Kamaci (2013) New Amperometric Cholesterol Biosensors Using Poly(ethyleneoxide) Conducting Polymers, Journal of Macromolecular 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, 1075–1084 CopyrightCTaylor & Francis Group, LLC
ISSN: 1060-1325 print / 1520-5738 online DOI: 10.1080/10601325.2013.821921
New Amperometric Cholesterol Biosensors Using
Poly(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, Turkey 2Ege University, Faculty of Science, Biochemistry Department, Bornova, Turkey 3Department of Chemistry, Selcuk University, Konya, Turkey
4Department 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 clinically important. In this study, two types of amperometric cholesterol biosensors were designed by physically entrapping cholesterol oxidase in 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 electrochemically and cholesterol oxidase was immobilized by entrapment during electropolymerization. The amperometric responses of the enzyme electrodes were measured by monitoring oxidation current of H2O2at+0.7 V in the absence of a mediator. Kinetic parameters, such
as 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 Km values, 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 a bioreceptor part and a transducer part (1). Bioreceptor of the biosensor is a biological molecular species or a living organism which is immobilized on a solid surface and utilizes a biochemical mechanism for recognition (2). The main advantage of a biosensor is its selectivity towards an analyte which is specific chemical or chemicals (3) in a complex sample. The signals recorded depend on the concentration of the analyte and they can be interpreted in terms of changes in resonance unit (4, 5), UV-Vis-IR absorption (6, 7), mass (8, 9), electrical (10, 11) and photoelectrical properties (12, 13). Enzyme biosensors have become increasingly important in clinical diagnostics, food industries and environmental control (14). Enzymes are expensive. Therefore, it is difficult and costly to separate them from the reaction mixture. However, the advantages
∗Address correspondence to: Huseyin Bekir Yildiz, Department
of 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 the product environment, enhanced stability and reduction in the 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 human blood can cause various clinical disorders, such as heart disease, coronary artery disease, arteriosclerosis, hyperten-sion, cerebral thrombosis, etc. (16). Moreover, cholesterol is also known to play an important role in the brain synapses and in the immune system. Therefore, the determination of cholesterol levels is of great importance in clinical analy-sis/diagnosis (17). Cholesterol oxidase is the enzyme which is mainly preferred and used in the cholesterol determina-tion studies. Cholesterol oxidase (E.C. 1.1.3.6) is a flavin adenosine dinucleotide (FAD)-containing enzyme and it catalyzes 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 chemical methods such as, colorimetric methods, chromatographic and 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
C C C O CH2 S CH3 CH2 O N H S N N N N H H H H n CH3OCH2CH2(OCH2CH2)nCH2 H m C C C O 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-known biochemical 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 the oxygen consumption or the hydrogen peroxide production can be monitored by using this technique.
After the first discovery of the conducting polymers, a new period has been started for the material and macro-molecular sciences. Conducting polymers have been used in many applications such as such as electrochromic materials (21) organic-based solar cells (22, 23), organic field effect transistors (24, 25), organic light-emitting diodes (26), drug release systems (27), rechargeable batteries (28) and immo-bilization of biomolecules (29). Conducting polymers are highly desirable in biosensor design because of their com-patibility with biological molecules, easy preparation, high reproducibility and electrochemical properties (30–32). Be-sides, they can have very well-organized molecular structure and therefore they can act as a three dimensional matrix for biomolecule immobilization (33–35). Immobilization of a biomolecule in electropolymerized films is a simple one-step method. In this physical entrapment, an appropri-ate potential is applied to the working electrode immersed in aqueous solution containing the biomolecule and the electropolymerizable monomer. This method enables
re-S CH2CH2 OH N H S N N N N O H H H H 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 with controlled thickness and morphology.
The present work describes two amperometric choles-terol biosensors which can determine cholescholes-terol sim-ple and fast by using poly(ethyleneoxide) conducting polymers. Cholesterol oxidase enzyme was entrapped in 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 their conducting 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). Although mediators enhance the sensitivity and selectivity (38–40), cholesterol sensors produced have enough sensitivity and selectivity towards the substrate; hence, a mediator was not used in this study. The biosensors were characterized in terms of several parameters such as operational and stor-age stabilities, kinetic parameters (Km and Imax), effects of pH and temperature.
2 Experimental
2.1 Material and Reagents
Cholesterol oxidase [E.C.1.1.3.6] (26.4 U/mg protein) from Pseudomonas fluorescens, cholesterol, sodium dodecyl sul-fate (SDS) and Triton X-100 were purchased from Sigma Aldrich and used with no further purification. Pyrrole
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Sch. 3. Schematic representation of immobilization of cholesterol oxidase in conducting copolymer matrices. (Color figure available
online.)
and hydrogen peroxide were supplied by Merck. Ethanol (Merck) was of analytical grade and used as received for preparing the cholesterol stock solution (0.05 M). Phos-phate buffer (pH= 7) for the electrosynthesis was prepared by dissolving 0.025 mol of Na2HPO4 (Fisher Scientific Company) 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), and 0.1 M KCl (Fisher Scientific Company) to provide ionic conductivity.
2.2 Instrumentation
Electrochemical measurements were carried out at ambient conditions (∼25◦C) in a cell equipped with Ag/AgCl ref-erence electrode (silver wire dipped in 4 M KCl saturated with 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 (The Netherlands) potentiostat via chronoamperometry. JEOL JSM-6400 model scanning electron microscope (SEM) was also used for the characterization of the biosensors.
2.3 Preparation of PEO-co-PPy/ChOx and CP-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
electrode was performed by applying+1.0 V at room tem-perature in a typical three-electrode cell containing CP coated platinum foil (1 cm2) as working, bare Pt as counter and Ag/Ag+ as reference electrodes. Electrolysis solu-tion for ChOx immobilizasolu-tion consists of ChOx (0.8 mg/ 10 mL), SDS (1.2 mg/mL), pyrrole (0.01 M) and phosphate buffer (10 mL, pH 7.0). For the immobilization of ChOx in 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) was put in a typical three electrode cell. The three electrode cell has the bare Pt as working and counter electrodes and a Ag/Ag+(4 M) reference electrode. Polymerization reac-tions for preparing of PEO-co-PPy/ChOx electrode were carried out by applying 1.0V. SDS is not only the support-ing electrolyte for the electrosynthesis, but it also enhances solubility of pyrrole (Py) in aqueous solutions, thanks to its ionic surfactant property. After electrolysis, the enzyme electrodes 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 when not 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 a clear solution. The stock solution was stored at+4◦C in the dark and consumed in 10 days. Triton X-100, the nonionic surfactant providing solubility of cholesterol in aqueous solutions, was added to the cholesterol solutions just be-fore the experiments. High Triton X-100 concentration can inhibit the activity of ChOx. The concentration range of 0.8–1.2% (v/v) was found to be suitable by Tan et al. (41). For all the experiments, this surfactant was added in the ratio of 1% (v/v) to the analyte solutions.
2.5 Measurements
All experiments were carried out at ambient conditions in an electrochemical cell containing 10 mL of phosphate buffer as previously described section of Material and reagents. After each run, the electrode was washed with distilled water. The biosensor was initially equilibrated in buffer and then the substrate was added to the medium. Current response due to cholesterol addition was recorded at the 150th second. The amperometric responses were mea-sured via monitoring oxidation current of H2O2at+0.7 V without using a mediator. Initially, the baseline current became constant, and then the analyte was added to the medium; the current immediately increased (response times were 2–3 s) and reached a steady state almost at the end of 150–200 s. Finally, the differences between these current values were recorded. During the experiments, the system was gently stirred.
2.6 Determination of Optimum pH and Optimum Temperature
The reaction temperature was changed between 10◦C and 60◦C while the cholesterol concentration was kept constant at 10 Km for every case. For pH optimization at 25◦C, (in order to be able to use these electrodes as biosensors) the pH of the reaction was altered between pH 4 and pH 11 while the cholesterol concentration was kept constant at 10 Km. In all experiments for both pH and temperature optimiza-tions, the enzyme activity determination experiments were performed via application of+0.7 V as previously described in the Measurements section. Relative enzyme activity was calculated by assigning the maximum value of activity as 100% 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 in 5 days throughout 30 days. In the investigations of both operational and storage stabilities, enzyme activity deter-mination was found via application of +0.7 V at pH 7.0 and 25◦C as previously described in the Measurements sec-tion. Substrate concentrations were kept at 10 Km and electrodes were stored in buffer solution at 4◦C when not in use. As done in determination of optimum pH experi-ments, relative enzyme activity was calculated by assigning the maximum values of activity as 100% in operational and storage 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 optimal thickness of the polymer film for making biosensors is very important. There are two reasons why this is important. One of them is that very thin polymeric films may be unable to protect the enzyme from the environmental effects. The other 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 the recognition element. The thickness was controlled by fixing the charge at which the maximum amperometric response was obtained. First, CP coated Pt electrodes were elec-tropolymerized with PPy depositing 1Q (0.0428 C-charge deposited in a minute), 2Q (0.0856 C), 3Q (0.128 C), and 4Q (0.171 C) and then the experiments were carried out by adding 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/cm2for 1Q, 2Q, 3Q and 4Q CP-co-PPy elec-trodes, respectively. In the case of PEO-co-PPy,after 1Q
New Cholesterol Biosensors Via Conducting Polymers
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(0.0428 C), 2Q (0.0856 C), 3Q (0.128 C), 4Q (0.171 C) and 5Q (0.214 C) deposition, the experiments were done by adding 5 mL hydrogen peroxide (0.222 mM in a total volume of 15 mL). The current responses were obtained as 21, 33, 45, 57 and 51μA/cm2for 1Q, 2Q, 3Q, 4Q and 5Q PEO-co-PPy electrodes, respectively. In summary, in all of the subsequent experiments, 3Q CP-co-PPy (nearly
80μm thick) and 4Q PEO-co-PPy (nearly 110 μm thick)
electrodes were utilized. The thickness of the polymers was estimated using the charge required for the film coating on the electrode surfaces. These values were checked with a micrometer after peeling off the polymer film from the electrode surface.
3.2 Enzyme Loading
Performance of the biosensor strongly depends on the amount of the immobilized enzymes since this would af-fect biosensor activity directly. To determine the optimal amount of enzyme loading, different biosensors contain-ing 0.4, 0.6, 0.8, 1.0 and 1.2 mg ChOx were prepared and the 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 loadincreas-ing and then reached a saturation point. The maximum responses were obtained with 0.8 mg pro-tein/10 ml and 1.0 mg propro-tein/10 ml for CP-co-PPy and PEO-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 mg protein/10 ml to obtain the highest sensitivity. The results for both sensors are shown in Figure 1.
3.3 Kinetic Parameters
Kinetic studies of the immobilized ChOx were performed at at constant temperature (25◦C) and pH (pH 7) while
vary-0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0 5 10 15 20 25 30 35 40 45 Current Response ( µ A/cm 2 ) Protein Concentration (mg/10 mL)
Fig. 1. Current responses containing different amounts of ChOx
in the presence of 10 mM cholesterol solution for CP-co-PPy/ChOx () and PEO-co-PPy/ChOx (◦) electrodes. (Color figure available online.)
ing cholesterol concentration. In the equilibrium model of Michaelis–Menten, the substrate binding step is as-sumed to be fast relative to the rate of breakdown of the enzyme–substrate complex (30). Kinetic parameters for the amperometric biosensors include the maximum reaction rate (Imax) of the enzymatic reaction and the Michaelis-Menten constant (Km) which is the equilibrium dissoci-ation constant for the complex. The Michaelis-Menten constant (Km), defines the affinity of enzyme toward its substrate corresponds to substrate concentration at 1/2 Imax (42). The lower the Km value means the higher its affinity against its substrate. The kinetic parameters, Imax and Km, were obtained from the Lineweaver-Burk plot which is a plot of 1/Vo against 1/[Substrate] for systems obeying the Michaelis-Menten equation. The graph be-ing linear can be extrapolated at anywhere approximat-ing to a saturatapproximat-ing substrate concentration, even if no experiment 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/ChOx and CP-co-PPy/ChOx electrodes were shown at Figure 2a and Figure 3a, respectively. Figure 2b and Figure 3b give the Lineweaver–Burk plots of these enzyme electrodes. The current responses that belong to minimum detectable con-centrations were obtained as 3.5 and 3.9μA/cm2for CP-co-PPy/ChOx and PEO-CP-co-PPy/ChOx electrodes respec-tively. Lower the Km value means higher affinity of enzyme for its substrate. Therefore, the reason for the lower Km value of PEO-co-PPy/ChOx electrode than that of CP-co-PPy/ChOx can be explained as that more ChOx was associated with cholesterol in PEO-co-PPy matrix. Sensi-tivity of an enzyme electrode can be described as Imax/Km ratio (43, 44) and sensitivities were calculated as 11.87 and 13.32 μA/mM.cm2 for CP-co-PPy/ChOx and PEO-co-PPy/ChOx enzyme electrodes, respectively (Table 1). It can be assumed that with small Km value and comparatively high sensitivity, the PEO-co-PPy/ChOx enzyme electrode showed better biosensor characteristics.
3.4 Effect of pH
The pH stabilities of the freshly prepared enzyme electrodes were determined via application of+0.7 V at different pHs ranging from 4 to 11 at 25◦C. Due to the denaturation of 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 13.32
CP-co-PPy/ChOx 61.26 5.16 11.87
0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.00 0.05 0.10 0.15 0.20 0.25 0.30 (b) 1/Current Response 1/Concentration y= 0.047 + 0.085x R2 = 0.994 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 16 18 20 (a) Current Response ( µ A/ cm 2 ) Concentration (mM)
Fig. 2. (a) Current response vs concentration and, (b) 1/Current
response vs. 1/concentration for the PEO-co-PPy/ChOx enzyme electrode (at pH 7 and 25◦C). (Color figure available online.)
0 2 4 6 8 10 12 14 0 5 10 15 20 25 30 35 40 45 50 (a) Current Response ( µ A/cm 2 ) Concentration (mM) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 (b) y= 0.005 + 0.112x R2 = 0.991 1/Current Response 1/Concentration
Fig. 3. (a) Current response vs concentration and, (b) 1/Current
response vs. 1/concentration for the CP-co-PPy/ChOx enzyme electrode (at pH 7 and 25◦C). (Color figure available online.)
0 2 4 6 8 10 12 0 20 40 60 80 100 120
Relative Enzyme Activity
pH
Fig. 4. Effect of pH on activity of cholesterol oxidase immobilized
in CP-co-PPy () and PEO-co-PPy (◦) matrices. (Color figure available online.)
The maximum activities were observed at pH 6.0 and 7.0 for CP-co-PPy and PEO-co-PPy matrices respectively. They are illustrated in Figure 4. Up to pH 11, although CP-co-PPy matrix lost 66% of its activity PEO-co-CP-co-PPy matrix lost only 21% of its activity. Moreover, PEO-co-PPy matrix was stable on the pH range between the pH 6 and 10 and hence it can be used reliably at high pH values for enzyme reactions. Moreover, due to the high stability of PEO-co-PPy/ChOx enzyme electrode on the pH range between pH 6 and 10, it has also the advantage in medical applications since the blood pH is 7.4. The optimum pHs were shifted towards the 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 concentrate protons, and this causes lowering the pH around the en-zyme. Therefore, the pH around the enzyme will be lower than that of the bulk phase from which the measurement of 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 zyme. The temperature stabilities of the freshly prepared en-zyme electrodes were determined via application of+0.7 V at 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 higher temperatures to prevent vaporization. As illustrated in Figure 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 responses as 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-CP-co-PPy matrices were calculated as 16.8 and 12.9 kJ/mol, respectively (Fig. 5b and 6b). The smaller
New Cholesterol Biosensors Via Conducting Polymers
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0 10 20 30 40 50 60 70 0 4 8 12 16 20 (a) Cur rent Response ( µ A/ cm 2 ) 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 ( current response, µ A/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 in PEO-co-PPy. (Color figure available online.)
Ea means that the ChOx enzyme entrapped in PEO-co-PPy matrix shows higher enzyme activity and the sensor exhibits higher affinity towards its substrate, which is in agreement with lower Km value.
3.6 Operational and Storage Stabilities of Enzyme Electrodes
Enzymes can easily lose their catalytic activity and dena-tured. Biomolecules like enzymes have limited stabilities especially when they are removed from their native areas and their stabilities and performances decrease due to im-mobilization. Therefore, operational and storage stability are important considerations for an immobilized enzyme. Operational and storage stabilities were shown in Figures 7 and 8, respectively. Operational stability of enzyme elec-trodes was tried to estimate the stability of elecelec-trodes in terms of 20 repetitive uses. PEO-co-PPy/ChOx electrode maintained an activity at 80% until the assay number 7 and exhibited a good stability upon the repetitive uses. After 6th assay, enzyme activity decreased and after 16th assay
0 10 20 30 40 50 60 70 0 10 20 30 40 50 (a) Temperature (°C) Current Response ( µ A/cm 2 ) 0.0030 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 (b) Ln ( current response, µ A/cm 2 ) 1/Temperature (K-1) y = 9.93 - 2017.59 X
Fig. 6. (a) Effect incubation temperature on activity of cholesterol
oxidase immobilized in CP-co-PPy matrix and, (b) Determination of 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 operational stability and retained 95% of its original activity until the assay number 9 and then kept 85% of its activity even af-ter 20th use. It was observed that although PEO-co-PPy
0 5 10 15 20 25 0 20 40 60 80 100 120
Relative Enzyme Activity
Assay Number
Fig. 7. Operational stabilities of CP-co-PPy/ChOx () and PEO-co-PPy/ChOx (◦) electrodes. (Color figure available online.)
matrix was likely to protect the enzyme better, as it is thicker than CP-co-PPy matrix, it is more vulnerable to environ-mental effects. Besides, the slight increase in the response of CP-co-PPy/ChOx electrode is related to the swelling of the polymer structure and it was speculated that swelling of the polymer may cause changing positions of the enzyme molecules in the polymer to increase the enzyme activity slightly. As known, due to physical entrapment, there is no bond between enzymes and the polymer in the polymer and when the polymer swells, the position changing for enzyme molecules can occur (45).
Storage stability of immobilized ChOx in PEO-co-PPy exhibited a 55% loss of its activity in 20 days and stayed constant until the end of its storage stability experiment. On the other hand, immobilized ChOx in CP-co-PPy matrix lost 15% of its activity in the first 15 days and then stayed constant with 85% of its original activity until the 30thday. In spite of having good enzyme protection against high temperature and pH, PEO-co-PPy/ChOx enzyme electrode showed worse storage stability than the CP-co-PPy/ChOx enzyme electrode. This can be explained that some amount of enzyme dropped to the solution from the matrix while being studied. Both electrodes have very good stabilities in the first 5 days and can be safely used in this period. Since immobilized ChOx in CP-co-PPy maintained 85% of its original activity after the 15thday and stayed constant up to the 30thday, it can also be used safely between the 15th and 30thdays with a very high activity.
3.7 Morphologies of Films
In order to determine the surface morphologies of polymer films, scanning electron microscopy (SEM) technique was used. Scanning electron micrographs of CP-co-PPy/ChOx and 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 were completely different compared to the films prepared in the absence of yeast cells. Cauliflower-like structure was
0 5 10 15 20 25 30 35 0 20 40 60 80 100 120
Relative Enzyme Activity
Days
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 matrix
with ChOx and (b) PEO-co-PPy matrix with ChOx.
noticeably damaged when yeast cells were entrapped in these matrices.
4 Conclusion
The redox enzyme, cholesterol oxidase, which catalyzes the oxidation ofβ-D-cholesterol to D-glucono-1,5-lactone and hydrogen peroxide, was immobilized in two differ-ent poly(ethyleneoxide) type matrices for the first time to
New Cholesterol Biosensors Via Conducting Polymers
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construct amperometric biosensors. The amperometric re-sponses of CP-co-PPy /ChOx and PEO-co-PPy/ChOx en-zyme electrodes were measured by monitoring oxidation current of H2O2 at+0.7 V in the absence of a mediator Kinetic parameters, operational and storage stabilities, op-timum temperature and pH were investigated for the ma-trices. PEO-co-PPy/ChOx electrode had smaller Km and Imax values and higher sensitivity when compared with Km and Vmax values of free enzyme and CP-co-PPy/tyrosinase electrode. The smaller Km value shows that PEO-co-PPy matrix provides a microenvironment which is more suit-able than that in the solution and CP-co-PPy matrix. It can be understood from higher sensivitiy that enzyme immo-bilized in PEO-co-PPy matrix has higher affinity towards its substrate than the enzyme immobilized in CP-co-PPy matrix does. In spite of having high temperature and pH stabilities, 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 enzyme dropped to solution from the matrix. Moreover, immobi-lization of cholesterol oxidase enzyme in these conducting polymer electrodes can be studied as an alternative biosen-sor fabrication for the determination of cholesterol amount in fruit juices without requiring sample pre-treatment and results show that this significant development can success-fully replace the classical methods. This study proves that conducting polymers; CP-co-PPy and PEO-co-PPy can be used as immobilization matrices for ChOx to produce am-perometric biosensors which can determine the cholesterol amount 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-BAP Grant Number 09-M-11) for the financial support of this research.
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