Construction of a Novel Glucose Biosensor Based on Covalent Immobilization of Glucose Oxidase on...

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Construction of a Novel Glucose Biosensor Based on CovalentImmobilization of Glucose Oxidase on Poly(glycidylmethacrylate-co-vinylferrocene)Mehmet Senel,a, c M. Fatih Abasıyanıkb, c*a Department of Chemistry, Faculty of Arts and Sciences, Fatih University, B.Cekmece, Istanbul 34500, Turkeyb Department of Genetics and Bioengineering, Faculty of Engineering, Fatih University, B.Cekmece, Istanbul 34500,Turkeyc Biotechnology Research Laboratory, Bionanotechnology Research Center, Fatih University, B.Cekmece, Istanbul 34500, TurkeyTel: þ902128663300 Ext:5642, Fax: þ902128663402*e-mail: [email protected]

Received: December 29, 2009Accepted: February 23, 2010

AbstractA novel glucose biosensor was constructed via direct covalent attachment of glucose oxidase onto epoxy groupcontaining polymeric electron transfer mediator, Poly(glycidyl methacrylate-co-vinylferrocene). A copolymer ofglycidyl methacrylate (GMA) and vinylferrocene (VFc) with different molar ratios has been prepared by free radicalcopolymerization. These copolymers have been utilized as polymeric mediators for amperometric glucose sensing.The catalytic electrochemistry of the enzyme electrode with the copolymer was investigated. Copolymer acts as anelectron transfer mediator between the redox center of Glucose oxidase (GOx) and the electrode. The stability,reusability, pH and temperature response of the biosensor as well as its kinetic parameter have also been studied.

Keywords: PGMA, Ferrocene, Glucose oxidase, Biosensors

DOI: 10.1002/elan.200900644

1. Introduction

Redox polymer-modified electrodes have been widelyinvestigated because of applications in sensors and biosen-sors to produce devices for reagentless sensing. Attachmentof bioactive molecules on transducer and electron transfercapacity of supporting film greatly influence the biosensorcharacteristics. Several enzymes such as horseradish perox-idase (HRP), glucose oxidase (GOx) and acetylcholinester-ase have been successfully immobilized and employed insensing applications [1 – 3]. However, electron transferefficiency of redox enzymes is poor in absence of mediator,because enzyme active sites are deeply embedded inside theprotein. The sensitivity of resulted biosensors can besignificantly improved by the addition of mediators insolution or in the matrices [4, 5]. Leakage is a main problemin the entrapment of mediator (low molecular weight)compounds. This problem can be resolved by covalentlinking of mediator with with high molecular weightcompounds before immobilization. Some biosensors havebeen reported through direct linking ferrocene with poly-(vinylferrocene-co-hydroxyethyl methacrylate) [6], poly-(N-acryloylpyrrolidine-co-vinylferrocene) [7], acrylamidecopolymers [8] and ferrocene-based polyamides [9].

Determination of the glucose concentration is importantin clinical diagnostics. Since Clark and Lyons reported thefirst enzyme electrode in 1962 [10] there has been consid-erable attention paid to developing novel biosensors for the

fast, reproducible, sensitive, and selective quantification ofglucose. There is increasing interest to develop an electro-chemical glucose biosensor by immobilizing enzymes on anelectrode surface. Majority of these studies used the enzymeglucose oxidase, which catalyzes the oxidation of glucose togluconic acid in the presence of oxygen and generateshydrogen peroxide. The detection of glucose by electro-chemical biosensors is based on the electrochemical oxida-tion of hydrogen peroxide generated by enzyme-catalyzedoxidation of glucose at anodic potentials (>þ 0.6 V vs. Ag/AgCl) [11, 12]. However, at this relatively high potential,there may be interferences from other oxidizable speciessuch as ascorbic acid, uric acid, and acetaminophen. Toavoid this problem, mediators were used to replace oxygenas the means of electron transfer [13 – 15]. Recently, due tofundamental interest in electron-transfer reaction betweenGOx and electrodes and from the view of long term stabilityof glucose sensors, several redox-active polymers have beenprepared and utilized as polymeric mediators [16 – 21].

Several researchers have tried to modify enzyme mole-cule itself. Ferrocene molecules as the electron relays werecovalently attached within the protein shell of GOx. Thismethod successfully applied this concept to several kinds ofoxidases and redox relays Degani and Heller [22, 23].Schumann et al. reported electron transfer between GOxand an electrode via ferrocene bound to the enzyme surfacewith flexible chains and stated that sufficient length wasrequired for the effective electron transfer [24]. GOx was

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Electroanalysis 2010, 22, No. 15, 1765 – 1771 � 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1765

reconstituted with ferrocene-modified Flavin adenine di-nucleotide (FAD) and FAD-removed GOx, and improvedelectrical communication between an electrode and thereconstituted enzyme [25]. Iwakura et al. reported anelectropolymerized film incorporating glucose oxidase andseveral kinds of mediator such as benzoquinone andferrocene derivatives [26].

In this research, we have constructed a novel glucosebiosensor based on a series of poly(glycidyl methacrylate-co-vinylferrocene) redox copolymers. This method is effec-tive in that the epoxy group can be easily introduced into thecopolymer at a desired density by adjusting the concen-tration of comonomer in the polymerization mixture. Thisability provides a one-step procedure for fabrication of aglucose biosensor. The application of the resulting enzymeelectrode for the amperometric detection of urea in aqueousmedium is described.

2. Experimental

2.1. Materials

Glycidyl methacrylate (GMA) and 2,2’-azobis(isobutyroni-trile) (AIBN) were purchased from Fluka and AcrosChemical Co. and used without further purification. Re-agent-grade vinylferrocene (VFc) was purchased fromAldrich Co. and used without purification. Glucose Oxidase(GOx) (EC 1.1.3.4) was obtained from Sigma. All otherchemicals were of analytical grade and were used withoutfurther purification.

2.2. Preparation and Characterization of Poly(GMA-co-VFc)

The redox polymer, having different compositions, wasprepared according to literature (Figure 1) [27]. A mixtureof VFc and GMA at a known molar ratio was injected into aPyrex tube, AIBN (1 mol%). The mixture was degassedwith argon and sealed under vacuum. After degassing, thetubes were placed in a constant-temperature bath set to70 8C. After two days, the reaction mixture was added drop-

wise to rapidly stirred diethyl ether to precipitate thecopolymer. Precipitated copolymer was washed with diethylether and reprecipitated two more times in this manner. Theprecipitated product was then dried under vacuum.

2.3. Preparation of Electrodes and Glucose Biosensors

To evaluate electrochemical properties, 10 mL of 1 wt%poly(GMA-co-VFc) in DMF was dropped onto a glassycarbon electrode. After drying at room temperature, thecyclic voltammogram (CV) of the copolymer was measuredin 100 mM phosphate-buffered 0.8% saline (PBS, pH 7.4).

Glucose Oxidase was immobilized by covalent attach-ment on a poly(GMA-co-VFc) coated glassy carbon elec-trode (GCE) (Figure 1). 1 wt% solution of poly(GMA-co-VFc) in DMF was dropped onto the electrode and dried inair. The copolymer film electrode with epoxy functionalitywas immersed in 100 mM phosphate buffer (pH 7.4) for 2 h,then transferred to fresh buffer containing GOx (2.0 mg/mL). Immobilization of GOx on the poly(GMA-co-VFc)film was carried out by continuously stirring the reactionmedium at 22 8C for 20 h. After this period, the electrodewas removed from the medium and washed with phosphatebuffer (100 mM, pH 7.4). The electrochemical properties(CVs) of poly(GMA-co-VFc) and enzyme electrodes weremeasured in pH 7.4 100 mM PBS at a scan rate 5 mV/s.

2.4. Protein Assay

The amounts of un-bounded protein on the electrodes weredetermined by Lowry method by using bovine serumalbumin as the standard [28]. The quantity of covalentlybounded protein was calculated by subtracting the recov-ered in the combined washings of the enzyme-electrodefrom the protein used for immobilization.

2.5. Electrochemical Measurements

Electrochemical measurements were performed using aCHI Model 842B electrochemical analyzer. A small glassy

Fig. 1. Immobilization of enzyme onto a PolyGMA-co-VFc) film electrode via the amine group.

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carbon working electrode (2 mm diameter), a platinum wirecounter electrode (0.2 mm diameter), an Ag/AgCl-saturat-ed KCl reference electrode and a conventional three-electrode electrochemical cell were purchased from CHInstruments.

All amperometric measurements were carried out atroom temperature in stirred solutions by applying thedesired potential and allowing the steady state current to bereached. Once prepared, the GOx electrodes were im-mersed in 10 ml pH 7.4 100 mM PBS solution and theamperometric response to the addition of a known amountof glucose solution was recorded. The data shown are theaverage of three measurements for each electrode.

3. Results and Discussion

3.1 Preparation and Characterization of Poly(GMA-co-VFc)

The most frequently used supports for covalent enzymeimmobilization in biotechnology have typically been ob-tained through activation of natural and synthetic polymerssuch as chitin, alginic acid, cellulose, acrylic and polyvinylalcohol [29 – 33]. Poly(GMA-co-VFc) with different mono-mer ratios were prepared from glycidyl methacrylate andvinylferrocene monomers as described in Section 2. Thepresent method was effective in that the reactive epoxygroup was readily introduced into the polymer supportwithout any modification. The epoxy group can bind proteinmolecules via their amine, thiol, hydroxyl and carboxylgroups in the pH range where the enzyme is stable and doesnot lose its activity. The chemical structure of poly(GMA-co-VFc) and the immobilization reaction via amine groupsare presented in Figure 1.

3.2. Cyclic Voltammograms of Poly(GMA-co-VFc) andGOx Immobilized Electrodes

The cyclic voltammograms of poly(GMA-co-VFc) wereobtained between 0.1 and 0.8 V in PBS after the copolymerwas cast onto a GCE. Figure 2 shows that the anodic andcathodic current of the copolymer film rise continuouslywith the number of potential scans until a distinct redoxcouple of Fc occurs and reaches steady state after 12 – 16cycles. After equilibrium was established, the peak Epa andEpc potentials remained constant. The steady state values forEpa and Epc for the cyclic voltammograms shown in Figure 2are 0.33 and 0.54 V, respectively.

Typical cyclic voltammograms of the electrode loadedwith copolymer in PBS are shown in Figure 3 for scan ratesfrom 1 to 20 mV/s. A linear correlation was obtainedbetween the anodic peak current (Ipa) and the square root ofthe scan rate (v1/2). This result indicates that chargepropagation in the polymer occurs by a diffusion-likeprocess, such as electron hopping between neighboringredox sites, and through counter-ion motion. Initially, the

ferric ion in the VFc units exists as a mixture of both thereduced (Fe(II)) and oxidized (Fe(III)) forms. Fe(II) isoxidized to Fe(III) during the forward scan, yielding anoxidation current peak. During the reverse scan, Fe(III) isreduced. The difference in the redox peaks increased withincreasing scan rate. The voltammetric behavior alsoindicates that ferrocene has been immobilized on thesurface of the glassy carbon electrode.

Figure 4 shows the current response of a poly(GMA-co-VFc)/GOx enzyme electrode in the absence and presence ofglucose. The increase in anodic current observed in thepresence of glucose indicates that the catalytic reactionenhances the oxidation current of the enzyme electrode in amanner similar to that reported previously [6]. During theenzymatic reaction, coenzyme FAD is converted to FADH2

in the presence of glucose, which subsequently reduces

Fig. 2. Cyclic voltammogram of polyGMA-co-VFc) between 1and 16 cycles in 0.1 M PBS pH 7.4) at a scan rate of 5 mV/s.

Fig. 3. Cyclic voltammograms of polyGMA-co-VFc) in 0.1 MPBS pH 7.4) at scan rates of 1, 2.5, 5, 7.5, 10, 15, 20 mV/s frominternal to external).

Biosensor Based on Immobilization of Glucose Oxidase

Electroanalysis 2010, 22, No. 15, 1765 – 1771 � 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.electroanalysis.wiley-vch.de 1767


ferrocinium ions to ferrocene. Hence oxidation of theregenerated ferrocene at applied potential causes anincrease in the anodic current when glucose is present.The more rapid electron transfer reaction from FADH2 tothe ferrocinium ions and the electron-hopping reactionbetween ferrocinium ions and ferrocene caused the de-crease of the reduction current of the ferrocinium ions. Thisresponse indicates that the poly(GMA-co-VFc)/GOx en-zyme electrode is useful for the detection of glucose.

3.3. Catalytic Current for Enzyme Electrode

Figure 5 shows the steady-state current response of glucosefor the various glucose oxidase electrodes with constantpotential of þ0.35 V vs. Ag/AgCl. As shown, during thesuccessive addition of 0.5 mM of glucose, a well definedresponse is observed. The catalytic current of the enzymeelectrodes increases with increasing glucose concentration.A plateau was observed for glucose concentration higherthan 10 mM that showing the characteristics of Michaelis-Menten kinetics, as seen for many amperometric enzymeelectrodes [34 – 35].

The Michaelis – Menten analysis results are summarizedin Table 1. Km

app and jmax values represent the linear responserange and the dynamic range of the sensors, respectively.These results do not show the intrinsic properties of theenzyme, but show the properties of each enzyme electrode.Figure 5 and Table 1 show that the catalytic currentresponse of the enzyme electrode changed with the copo-lymer composition. The catalytic response of the enzymeelectrode reaches a maximum at a VFc ratio of 0.4.Increasing the concentration of the VFc unit increases theOx concentration near GOx and decreases the hydro-philicity of the copolymer. Decreased hydrophilicity of thecopolymers may lead to decreases in the penetration ofglucose into the enzyme electrodes, which would decreasethe catalytic current [6]. Also, increases in the concentrationof GMA increase the amount of bound GOx on theelectrode, as seen from inset of Figure 5. Maximal enzymebinding was observed for a VFc composition ratio of 0.2.Minimal enzyme binding occurred at a VFc compositionratio of 0.8. The enzyme binding capacity was similar for

Fig. 4. Cyclic voltammograms of polyGMA-co-VFc)/GOx in0.1 M PBS pH 7.4) in the a) absence, b) presence of glucose at ascan rate of 5 mV/s.

Fig. 5. Dependence of current on glucose concentration for enzyme electrodes. The current was measured at þ0.35 V vs. Ag/AgClafter applying the potential for 600 s. Inset: Immobilization efficiency of copolymer-electrode.




composition ratios of 0.4 and 0.6. As a consequence, thecatalytic current response was not directly related to theamount of enzyme bound to the electrode.

3.4. Response of Enzyme Electrode to Glucose at FixedPotential

Figure 6 shows the catalytic current response of the enzymeelectrode toward the stepwise increases of glucose concen-tration under optimized conditions. The electrode waspolarized atþ0.35 V in PBS to reach a steady-state current.Just after the addition of glucose, the time required to reach95% of the maximum steady-state current was less than 5 s,which indicates a fast response. This rapid response wasmainly due to the existence of the ferrocene group in thecopolymer as a mediator of electron transfer [7]. Theamperometric response increased linearly with increasingglucose concentration in the range from 0.5 to 6 mM. Thelimit of detection was 3.0 mM at a signal to noise ratio of 3.The curvature from the initial straight line showed thecharacteristics of Michaelis-Menten kinetics. The apparentMichaelis-Menten constant (KM

app), which gives an indica-tion of the enzyme-substrate kinetics for the biosensor, iscalculated to be 12 mM using the Lineweaver-Burk equa-tion [36]. This KM

app value was much lower than those of

native GOx in solution (27.0 mM) [37], immobilized GOx insol-gel organic-inorganic hybrid material (20 mM) [38], sol-gel/chitosan composite (21 mM) [39] and polypyrrole(25.3 mM) [40]. The low KM

app value indicated that theenzyme immobilized in poly(GMA-co-VFc)0.4 retained itsactivity and that there was a low diffusion barrier. Inparticular, the proposed method avoids using electrontransfer mediators similar to those needed for traditionalenzyme-based biosensors. As a result, the electrodesdeveloped in this research provide a means of reagentlessglucose biosensing.

3.5. Effect of the pH

The pH value is another variable which affects the responseof the enzyme electrodes. The pH dependence of theresponse of the enzyme electrode was determined using apH 7,4 100 mM PBS, and the pH varied between 5.0 and 9.0.Figure 7 shows that the maximum activity was obtained atpH 7.5. For each point in Figure 7, a new enzyme electrodewas prepared in order to eliminate the errors that mightarise from the reuse the enzyme-loaded copolymer.

3.6. Effect of the Temperature

The effect of temperature of the buffer solution on theresponse of the poly(GMA-co-VFc) mediated glucosebiosensor system was studied in the range of 20 – 60 8C(Figure 8). It has seen that the amperometric responseinitially increases with the temperature and decrease later.The response reached a maximum at about 45 8C. Thedecrease of the response after 50 8C may be attributed to thethermal inactivation of the enzyme.

Table 1. Apparent Michaelis – Menten constants, maximum cur-rent densities for enzyme electrodes.

Abbreviation Kmapp (mM) jmax (nA)

Poly(GMA-co-VFc)(0.2) 12.0 287Poly(GMA-co-VFc)(0.4) 10.1 226Poly(GMA-co-VFc)(0.6) 7.1 150Poly(GMA-co-VFc)(0.8) 11.6 163

Fig. 6. Amperometric response of enzyme electrodes to succes-sive glucose injections at an applied potential þ0.35 V in stirred0.1 M PBS. The inset shows the calibration curve.

Fig. 7. Effect of pH on GOx immobilized electrodes. Datacollected with freshly prepared enzyme electrodes refer to theaverage of three experiments; ca. 25 8C).




3.7. Selectivity, Sample Analysis and Stability

To demonstrate the practical use of the prepared enzymeelectrode human plasma samples were assayed. First, freshplasma samples were analyzed in the local hospital, then, thesamples were re-assayed with the enzyme electrode. Aplasma sample (1.0 mL) was added into 5 mL PBS (pH 7.4),and the response was obtained at constant potential(þ0.35 V). The glucose content of the blood plasma canthen be calculated from the calibration plot of the enzymeelectrode. The results in Table 2 are satisfactory and agreeclosely with those by the biochemical analyzer in hospital.

The selectivity of the enzyme electrode for glucose couldbe interfered by electroactive compounds in the analysis of

plasma samples. The interference tests were carried withthree common interfering compounds: ascorbic acid, uricacid and acetaminophen at the applied potential ofþ0.35 Vversus Ag/AgCl in PBS (pH 7.4) containing 5 mM glucose,respectively. The current of 0.5 mM uric acid was nearlyeliminated, while 0.8 nA for 0.1 mM ascorbic acid and0.6 nA for 0.1 mM acetaminophen, indicating that nonoticeable changes in current were detected for theinterferences in glucose solution. Compared to the highsensitivity of the result glucose biosensor, the interferencecurrent can be negligible.

The response of the GOx electrode was measured of itsresponse to 10 mM glucose for a period of 60 days. Theresults of 30 measurements during this period is plotted inFigure 9. The first 20 days owing to the desorption ofenzyme, that is weakly entrapped in the copolymer. Theactivity remains constant thereafter, indicating very goodlong – term stability of the electrode.

4. Conclusions

In this work, a new amperometric biosensor for thedetermination of glucose was developed and characterized.The catalytic current responses are highly sensitive, increas-ing almost linearly with an increase in glucose concentra-tions up to 10 mM. The catalytic response of the enzymeelectrode achieves maximum with increasing the VFccomposition up to a 0.4 ratio. The amperometric enzymeelectrode developed in this study provided linearity to theglucose in the 0.1 – 6.0 mM glucose concentration range.This new enzyme electrode, whose characteristics aredescribed above, appears to be simple to prepare, fast torespond, inexpensive and reasonably sensitive.

Acknowledgements

This research was supported by grants from T. R. PrimeMinistry State Planning Organization and Fatih UniversityResearch Support Office (P50090801-1).

References

[1] P. C. Pandey, S. Upadhyay, I. Tiwari, V. S. Tripathi, Electro-analysis 1999, 11, 1251.

[2] P. C. Pandey, S. Upadhyay, I. Tiwari, V. S. Tripathi, Sens.Actuators B 2001, 72, 224.

Fig. 8. Effect of temperature on GOx immobilized electrodes.Data collected with freshly prepared enzyme electrodes refer tothe average of three experiments pH 7.0).

Fig. 9. Long-time stability of GOx immobilized electrode. Theamperometric responses of these enzyme electrodes are regularlychecked during 60 days pH 7.0; ca. 25 8C).

Table 2. Glucoe content in human blood samples.

Sample number Determinedby hospital(mM)

Measuredby biosensor(mM)

Bias(mM)

1 2.26 2.35 þ0.092 3.64 3.75 þ0.113 4.42 4.58 þ0.16




[3] P. C. Pandey, S. Upadhyay, H. C. Pathak, I. Tiwari, Sens.Actuators B 2000, 62, 109.

[4] Yu, S. Liu, H. X. Ju, Biosens. Bioelectron. 2003, 19, 401.[5] P. C. Pandey, S. Upadhyay, I. Tiwari, S. Sharma, Electro-

analysis 2001, 13, 1519.[6] T. Saito, M. Watanabe, React. Funct. Polym. 1998, 37, 263.[7] S. Koide, K. Yokoyama, J. Elect. Anal. Chem. 1999, 468, 193.[8] N. Kuramoto, Y. Shishido, K. Nagai, Macromol. Rap.

Commun. 1994, 15, 441.[9] N. Kuramoto, Y. Shishido, Polymer 1998, 3, 669.

[10] L. C. Clark, J. C. Lyons, Ann. N.Y. Acad. Sci. 1962, 102, 29.[11] R. M. Ianniello, A. M. Yacynyeh, Anal. Chem. 1981, 53, 2090.[12] J. Wang, N. Naser, L. Angnes, W. Hui, L. Chen, Anal. Chem.

1992, 64, 1285.[13] A. E. G Cass, G. Davis, G. D. Francis, H. A. O. Hill, W. J.

Aston, I. J. Higgins, E. V. Plotkin, L. D. L. Scott, A. P. F.Turner, Anal. Chem. 1984, 56, 667.

[14] J. M. Dicks, W. J. Aston, G. Davis, A. P. F. Turner, Anal.Chim. Acta 1986, 182, 103.

[15] J. Wang, L. Wu, W. Lu, R. Li, J. Sanchez, Anal. Chim. Acta1990, 228, 251.

[16] Y. Himuro, M. Takai, K. Ishihara, Trans. Mater. Res. Soc. Jpn.2005, 30, 417.

[17] J. D. Qiu, W. M. Zhou, J. Guo, R. Wang, R. P. Liang, Anal.Biochem. 2009, 385, 264.

[18] Q. Sheng, J. Zheng, Biosens., Bioelectronics 2009, 24, 1621.[19] J. D. Qui, M. Q. Deng, R. P. Liang, M. Xiong, Sens. Actuators

B 2008, 135, 181.[20] G. Ren, X. Xu, Q. Liu, J. Cheng, X. Yuan, L. Wu, Y. Wan,

React. Funct. Polym. 2006, 66, 1559.[21] V. K. Gade, D. J. Shirale, P. D. Gaikwad, P. A. Savale, K. P.

Kakde, H. J. Kharat, M. D. Shirsat, React. Funct. Polym.2006, 66, 1420.

[22] Y. Degani, A. Heller, J. Phys. Chem. 1987, 91, 1285.[23] Y. Degani, A. Heller, J. Am. Chem. Soc. 1988, 110, 2615.[24] W. Schumann, T. J.Ohara, H. L. Schmidt, A. Heller, J. Am.

Chem. Soc. 1991, 113, 1394.[25] A. Riklin, K. Katz, I. Willner, A. Stocker, A. F. Buchkmann,

Nature London 1995, 376, 672.[26] C. Iwakura, Y. Kajiya, H. Yoneyama, J. Chem. Soc. Chem.

Commun. 1988, 1019.[27] F. Dursun, S. K. Ozoner, A. Demirci, M. Gorur, B. Keskinler,

F. Yilmaz, E. Erhan, Biomacromolecules, in press.[28] O. Lowry, N. J. Rosebrough, AL. Farr, R. J. Randall, J. Biol.

Chem. 1951, 193, 263.[29] S. Akgçl, Y. Kacar, A. Denizli, M. Y. Arıca. Food Chem.

2001, 74, 281.[30] K. Sarıoglu, N. Demir, J. Acar, M. Mutlu. J. Food Eng. 2001,

47, 271.[31] G. Spagna, R. N. Barbagallo, P. G. Pifferi, R. M. Blanco, J. M.

Guisan. J. Mol. Catal. B 2000, 11, 63.[32] E. Sahmetlioglu, H. Y�r�k, L. Toppare, I. Cianga, Y. Yagci,

React. Funct. Polym. 2006, 66, 365.[33] T. Bahar, A. Tuncel, React. Funct. Polym. 2004, 61, 203.[34] J. D. Qiu, W. M. Zhou, J. Guo, R. Wang, R. P. Liang, Anal.

Biochem. 2009, 385, 264.[35] J. D. Qui, M. Q. Deng, R. P. Liang, M. Xiong, Sens. Actuators

B 2008, 135, 181.[36] R. A. Kamin, G. S. Wilson, Anal. Chem. 1980, 52, 1198.[37] M. J. Rogers, K. G. Brandt, Biochemistry 1971, 10, 4624.[38] B. Q. Wang, B. Li, Q. Deng, S. Dong, Anal. Chem. 1998, 70,

3170.[39] X. Chen, J. B. Jia, S. J. Dong, Electroanalysis 2003, 15, 608.[40] J. C. Vidal, E. Garcia, J. R. Castillo, Biosens. Bioelectron.

1998, 13, 371.




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