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Thermoresponsive Poly(N-isopropylacrylamide) Gel for Immobilization of Laccase on Indium Tin Oxide Electrodes Maciej Klis,† Marcin Karbarz,† Zbigniew Stojek,† Jerzy Rogalski,‡ and Renata Bilewicz*,† Faculty of Chemistry, UniVersity of Warsaw, Pasteura 1, 02-093 Warsaw, Poland, and Department of Biochemistry, Maria Curie Sklodowska UniVersity, Sklodowskiej Sq 3, Lublin 20-031, Poland ReceiVed: October 23, 2008; ReVised Manuscript ReceiVed: January 20, 2009
We report on the properties of hydrogel matrix for the immobilization of laccase on conductive supports. The poly(N-isopropylacrylamide) gel is attached firmly to the indium-tin oxide (ITO) electrode, following its silanization with dimethylethoxyvinylsilane. The enzyme entrapped in the gel structure remained active longer than in the solution, and its redox and catalytic properties could be investigated by voltammetric methods. The reduction signals of the active sites, T1 and T2, of the Cerrena unicolor laccase were determined to be 0.79 and 0.38 V, respectively. The laccase catalytic activity toward oxygen in poly(N-isopropylacrylamide) was found to depend strongly on temperature. Reversible swelling/shrinking of the matrix was studied at 30 and 35 °C. Shrinking of the gel at higher temperature considerably decreased the efficiency of the catalytic reaction, however, interestingly, did not lead to irreversible changes in the enzyme structure. At temperatures below that corresponding to volume phase transition, the catalytic properties of the film were fully restored. High catalytic efficiency of the gel immobilized enzyme made it possible to employ the gel covered electrode for monitoring oxygen in solutions. 1. Introduction Among important applications of redox enzymes immobilized on conductive supports are electrochemical sensors and electrode materials that can be applied in biofuel cells. It has been recognized for many years that the enzymatic reduction of oxygen to water could provide an alternative to noble metal electrocatalysis for fuel cell cathodes. Laccase (EC 1.10.3.2) is one of the few enzymes that reduce O2 directly to H2O in a four-electron-transfer process,1 which makes it possible to use the enzyme as the component of a biofuel cell cathode2-10 or oxygen biosensor.11 Simultaneously with dioxygen reduction, oxidizing of substrate takes place.1 Laccase is an enzyme of wide specificity, capable of oxidizing numerous organic (e.g., phenolic and related) compounds and inorganic substrates.12 Due to their low substrate specificity, laccases comprise the group of enzymes with significant applications.13 Apart from biofuel cells and biosensors,14-16 laccases have practical applications ranging from pulp and paper industry to bioremediation and organic synthesis.13 In spite of the fact that laccases were found in fungi,17 plants,18 bacteria,19 and even insects,20 mainly the fungal enzymes are employed in biotechnological processes because of cheap production of pure preparations. Fungal Cerrena unicolor (C. unicolor) laccase used in present work is a glycoprotein (from 17 up to 24% carbohydrates) with a molecular weight in the range from 55 to 79 kDa.21,22 The enzyme active site contains four copper atoms of types I (blue copper), II, and III (two coppers),1 which play different roles in the enzymatic process. Substrates are oxidized at the T1 site, the primary electron acceptor of the enzyme, and the electrons are transferred through an intramolecular electron transfer (IET) * To whom correspondence should be addressed. Tel.: +48 22 8220211ext. 345. Fax: +48 22 8224889. E-mail:
[email protected]. † University of Warsaw. ‡ Maria Curie Sklodowska University.
mechanism via a His-Cys-His bridge to the T2/T3 cluster, where molecular oxygen is reduced.1 The aim of this paper is to study the properties of laccase molecules entrapped in poly(N-isopropylacrylamide) gel anchored at indium-tin oxide (ITO) electrodes. The polymeric hydrogels are cross-linked polymer networks, which are filled with water. The content of the liquid in hydrogels is usually higher than 95%; nevertheless, these materials have properties of liquids and solids. The unique internal structure makes polymeric hydrogels useful as a matrix for the immobilization of enzymes.23-25 Electronic communication between the electrode and the copper sites of laccase achieved in this matrix allowed to estimate the formal potential of the enzyme. Addition of common mediators lead to improvement of the catalytic efficiency of the immobilized enzyme toward oxygen reduction. It opened the possibility to apply the modified electrode for monitoring oxygen level in solutions. The phase transition of the matrix is an important property of the gel from the viewpoint of the efficiency of catalytic reaction. Increasing the temperature over the phase transition temperature leads to the shrinking of the matrix together with considerable reduction of the catalytic efficiency of the immobilized laccase toward oxygen while reswelling below this temperature returns the catalytic effect to its original value. 2. Experimental Section Chemicals. Polymer constituents, N-isopropylacrylamide (NIPA, 97%), N,N′-methylenebisacrylamide (BIS, 99%), ammonium persulphate (APS, 99.99%), and N,N,N′,N′-tetramethylethylenediamine (TEMED, 99.5%), were purchased from Aldrich. Dimethylethoxyvinylsilane (C6H14SiO, 98%), as a 10% solution in toluene, was used to link the hydrogel thin film with the electrode surface. The electroactive species, 1,1′-ferrocenedimethanol (Fc(MeOH)2, 98%), was purchased from Aldrich; 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS, 98%) was bought from Sigma. Buffer components, Na2HPO4 and
10.1021/jp8094159 CCC: $40.75 2009 American Chemical Society Published on Web 04/06/2009
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Figure 1. Scheme of the synthesis of NIPA hydrogel anchored to the ITO surface.
citrate acid, were from POCh. All chemicals were used as received except for NIPA, which was recrystallized twice from the benzene/hexane mixture (90:10 (v/v)). All solutions were prepared using high-purity water obtained from a Milli-Q Plus/ Millipore purification system (conductivity of water, 0.056 mS · cm-1). C. unicolor C-139 was obtained from the culture collection of the Regensberg University and deposited in the fungal collection of the Department of Biochemistry (Maria CurieSklodowska University, Lublin, Poland) under the strain number 139. Laccase from the fermentor scale cultivation was obtained according to an already reported procedure after ion exchange chromatography on DEAE-Sepharose (fast flow)26 and lyophilized on Labconco (Kansas City, MO, FreeZone Lyophilizer). Enzyme activity was measured spectrophotometrically with syringaldazine as the substrate for laccase.27 The protein content was determined according to Bradford with bovine albumin as the standard.28 The concentration of isolated and frozen (-18 °C) enzyme was Clacc ) 178 mg/L and activity 11.16 U/mL. After lyophilizing the laccase activity dissolved in 1 mL of water was 68.99 U/mL and Clacc ) 1180 mg/L. Indium Tin Oxide Electrodes. Glass slabs coated with ITO were purchased from Balzers. First, the electrodes were cleaned with acetone. Then they were immersed into a solution of H2O2 (3%) for 20 min under ultrasonication. Finally, they were rinsed a few times with water and dried. Synthesis and Anchoring of NIPA Hydrogels with Immobilized Laccase at ITO Surface. Prior to the attachment of the poly(N-isopropylacrylamide) gel to the surface of ITO electrodes, the surface was treated with a 10% toluene solution of dimethylethoxyvinylsilane to introduce the vinyl groups onto the ITO surface. Then the electrodes were washed with pure toluene to remove physisorbed silanes. Next, the ITO slabs were partly immersed in the water solution (pregel solution) perpendicular to each other. The pregel solution was previously degassed. It contained NIPA and BIS at concentrations of 693
and 7 mM, respectively. The NIPA hydrogel polymerization was initiated and accelerated by APS (1.88 mM) and TEMED (32 mM). After 20 h of polymerization, a thin hydrogel film/ layer ca. 500 µm thick and bound to the ITO surface was obtained. The scheme of the synthesis is shown in Figure 1. To immobilize laccase in the gel matrix, the enzyme was added to the pregel solution. Electrochemistry. Cyclic voltammetry were performed using the CHI 750B potentiostat (CH Instrument, Austin, TX) in a three-electrode arrangement, with a saturated Ag/AgCl electrode as the reference electrode and platinum foil as the counter electrode. The working electrodes were the ITO electrodes modified according to procedures described above. All measurements were performed in McIlvaine buffer solution (0.1 M citric acid + 0.2 M NaH2PO4) at pH 5.2, degassed with pure argon or saturated with medical oxygen. Spectrophotometry. In spectroelectrochemical experiments a model LAMBDA-25, UV-vis, Perkin-Elmer spectrometer was employed. 3. Results Figure 2 represents the scheme of the electrode, with the enzyme mechanically entrapped in the polymer matrix. It cannot be precluded, however, that some of the laccase molecules are covalently bound during the free radical polymerization process. Microscopic image of the electrode cross-section shows that the polymer layer is 0.5 mm thick. To confirm whether the enzyme in the gel is immobilized permanently, a spectrophotometric measurement was performed. The electrode was immersed in 10 mL of buffer solution for 24 h, and after that the solution was checked for the presence of laccase with a spectrophotometric ABTS common test. To monitor the laccase diffusion from the electrode layer to the bulk solution, 0.9 mL samples of this solution were mixed with 0.1 mL of 1 mM ABTS at time zero and the absorbance at 420
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Figure 2. Scheme of ITO electrode modified with poly(N-isopropylacrylamide) gel containing laccase. Microscopic picture of the electrode crosssection.
Figure 3. Spectrophotometric test of the electrode stability. Dependence of absorbance on time recorded for the solutions remaining after the electrode immersions in 1 mM ABTS solutions, λ ) 420 nm.
nm of the samples was recorded during 10 min. After the measurement, the electrode was again immersed in a 10 mL volume of fresh buffer for 24 h, and the experiment was repeated the next day. The dependence of absorbance on time is presented in Figure 3. After the first 24 h immersion (day 1), the solution contained the released enzyme, which oxidized in several seconds the ABTS present in the sample. After the next 24 h immersion (day 2), removal of some laccase from the electrode to the solution could be still observed, however, considerably less than during the first 24 h. After the third day of immersion, oxidation of ABTS was not seen anymore, indicating that all untrapped laccase had been earlier removed from the gel layer, and after that, the amount of enzyme in the film became constant. Such washed, stable electrodes were used in all further experiments. Catalytic activity of the electrode toward oxygen reduction proves the presence of laccase in the film. Figure 4 allows comparison of the voltammograms recorded using electrodes covered with pure NIPA gel and with gel containing laccase. When the ITO-gel electrode without enzyme is used, the reduction of oxygen takes place at 0 V vs NHE. When the ITO-gel/laccase electrode is employed, two additional reduction signals, c1 and c2, at 0.38 and 0.79 V vs NHE are seen. The peak at more positive potential can be attributed to one-electron transformation of the T1 copper site of laccase, whereas the low-potential process is most likely related to the transformation of the T2 site cluster of the enzyme. The reoxidation peak is seen at 0.9 V versus NHE. The fungal enzymes have the highest redox potentials of the T1 site;29 the values of the redox potential of the T1 site in different laccases have been found to be between 0.43 and 0.79 V vs NHE.29 This is one of the most important characteristics
Figure 4. Voltammograms recorded using ITO electrode modified with poly(N-isopropylacrylamide) gel with C. unicolor laccase (solid line) and without enzyme (broken line). Oxygen-saturated McIlvaine buffer solution, pH 5.2. Scan rate: 10 mV · s-1.
of laccase. The T1 site redox potential is a crucial parameter for biofuel cell applications since it determines the voltage of the device.30,31 High redox potential laccases are, therefore, of special interest in biotechnology. The T1 potential value, 0.79 V vs NHE, stays in good agreement with our previous study on laccase from Cerrena basidiomycetes (ca. 0.75 V vs NHE)32 and is confirmed by the values of the steady-state potential of C. unicolor laccase-modified spectrographic graphite electrode. The half-wave potential of O2 electroreduction on this electrode was found to be 0.80 and 0.79 V vs NHE, respectively.33 At the T2/T3 cluster the reduction of oxygen to water takes place.1 The reduction potential of the T2 center was found to be close to 0.40 V vs NHE, as was reported for other blue multicopper oxidases: fungal laccase,34 tree laccase,35 bilirubin oxidase,36 and ascorbate oxidase37sin line with the value obtained in this study. Slight decrease of the signal is attributed to the catalytic reduction of oxygen at the T2/T3 cluster, as the experiment was performed in aerobic conditions. However, this process is not efficient, probably because the enzyme is in the “resting fully oxidized” form. In this form, the T1 site can still be reduced by the electrode, but the electron transfer to the trinuclear cluster is too slow to be catalytically effective.29 To increase the catalytic efficiency of the gel-immobilized laccase, a mediator has to be added to the gel-laccase film in order to help in the regeneration of the reduced Cu+ form of the enzyme. Introduction of the mediator can be done by simple immersion of the electrode in the solution of mediator (0.1 mM) for about 45 min (Figure 5). Following 45 min of cyclic in the 1,1′-ferrocenedimethanol solution, the voltammetric peaks of Fc(MeOH)2 attain constant peak current value. This means that
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Figure 5. Voltammograms recorded using ITO electrode modified with poly(N-isopropylacrylamide) gel immersed in 10-4 M 1,1′-ferrocenedimethanol. Scan rate: 20 mV · s-1. Increasing signals obtained in 0-60 min time period. Inset: dependence of reduction peak current on time of immersion.
the gel is fully saturated with mediator. The diffusion process backward, to the solution, is also possible. This prevents the shortening of a lifetime of laccase embedded into the gel, since radical species produced in the catalytic reaction may denaturate the protein, if they remain in close vicinity of the reaction site.38,39 Diffusion coefficient of Fc(MeOH)2 in a similar film is depressed only by 19% compared to its value in water.40 Thus, in the presence of the mediator laccase becomes a highly efficient catalyst in the gel matrix (Figure 6A,B). Using two different mediators, namely, Fc(MeOH)2 (Figure 6A) and ABTS (Figure 6B), the potential of the catalytic oxygen reduction can be changed from 0.1 to 0.4 V, respectively. The Icat./Idiff values are 10.6 and 25,3, respectively. Current density for the ABTS mediated process is 38 µA · cm-2 (10-4 M ABTS, oxygen saturated buffer, pH 5.2). The dependence of the catalytic current on the mediator concentration was studied (Figure 7). For each measurement, a freshly prepared ABTS solution saturated with oxygen was employed. Between the experiments, the electrode was stored in buffer solution, and then directly before the experiment, the NIPA gel layer was saturated with ABTS2- for approximately 45 min. LineweaverBurk plots (Figure 7 inset) were used for the determination of the kinetic parameters for the enzyme in the gel matrix. The apparent Michaelis constant for ABTS (Kmapp) and the maximal current (Imax) for the enzyme electrode were estimated using the equation:41
Figure 6. (A) Voltammograms recorded using ITO electrode modified with poly(N-isopropylacrylamide) gel with C. unicolor laccase: 10-4 M 1,1′-ferrocenedimethanol in McIlvaine buffer solution, pH 5.2, saturated with oxygen (solid line) and argon (broken line). Scan rate: 1 mV · s-1. (B) Voltammograms recorded using ITO electrode modified with poly(N-isopropylacrylamide) gel with C. unicolor laccase: 10-4 M ABTS in McIlvaine buffer solution, pH 5.2, saturated with oxygen (solid line) and argon (dotted line) for scan rate of 1 mV · s-1; (dashed line) oxygen saturated buffer for scan rate of 50 mV · s-1.
1/I0 ) (Kmapp /Imax)(1/[S]) + 1/Imax where I0 is the catalytic wave current and [S] is the concentration of ABTS. Kmapp was found to be 0.50 ( 0.036 mM, and Imax was 31 µA, yielding jmax ) 62 µA · cm-2. The Km value is similar to the one reported for native C. unicolor laccase (ca. 0.45 mM).42 Although usually Km values of high redox potential laccases toward a suitable substrate as ABTS do not exceed 0.1 mM, conformational changes and partial denaturation of the lyophilized preparation may explain the higher Km value. Since the catalytic wave current depends on the concentrations of the enzyme substrates, we employed the device to monitor the oxygen concentration in the solution. The reaction of catalytic oxygen reduction takes place in the air-saturated buffer (Figure 8A). When the solution is deoxygenated slowly by passing argon, the catalytic wave decreases and turns into a common
Figure 7. Voltammograms recorded using ITO electrode modified with poly(N-isopropylacrylamide) gel with C. unicolor laccase: oxygensaturated McIlvaine buffer solution, pH 5.2, containing ABTS in different concentrations. Scan rate: 5 mV · s-1. Inset: Lineweaver-Burk plot of laccase reaction with ABTS.
peak-shaped voltammogram corresponding to the Fc(MeOH)2 compound processes. The dependence of the current signal on time of deoxidation is shown in Figure 8B. These measurements show the utility of the device as an oxygen sensing tool in solutions containing oxygen at 10-4-10-3 M level. No attempts to measure lower concentrations were made. The electrode can be used both in the voltammetric and amperometric regime. The electrode
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Figure 9. Voltammograms recorded using an ITO electrode modified with poly(N-isopropylacrylamide) gel with immobilized laccase: 10-4 M 1,1′-ferrocenedimethanol in buffer solution saturated with oxygen, pH 5.2, at temperatures below (solid black line, 30 °C) and above (dashed line, 35 °C) the volume-phase-transition temperature. Situation after reswelling: dotted line, 30 °C. Scan rate: 5 mV · s-1.
4. Conclusions
Figure 8. (A) Voltammograms recorded using ITO electrode modified with poly(N-isopropylacrylamide) gel with C. unicolor laccase: 10-4 M ABTS in buffer solution, pH 5,2. Slow deoxidation of air-saturated solution with argon causes the drop of mediator reduction signal. Scan rate: 5 mV · s-1. (B) Dependence of reduction peak current on the time of deoxidation.
response is fast and the stable signal is obtained in few minutes. For five measurements in the same solution the repeatability was higher than 95%. It is known that NIPA gels undergo volume phase transition in water solutions in response to changes in temperature for both for gels in free samples43,44 and in thin layers attached to surfaces.40 The influence of the volume phase transition on the efficiency of the catalytic reaction was therefore checked. The gel attached to the electrode surface exhibited a sharp change in the volume when the temperature exceeded 34 °C, similarly to the behavior of the free gel sample grown in a capillary. The voltamograms shown in Figure 9 were obtained at a temperature below (solid line, 30 °C) and above (dashed line, 35 °C) the gel volume-phase-transition temperature. The height of the catalytic wave obtained at temperature 35 °C is ca. 1 order of magnitude lower than that of the wave recorded at temperature 30 °C. In addition, the catalytic wave obtained after the reswelling (dotted line, 30 °C) is of very similar height to the one recorded before volume phase transition. These results clearly show that shrinking of the gel considerably decreases the efficiency of the catalytic reaction, however, interestingly, does not lead to irreversible changes of the enzyme structure. Moving to conditions below phase transition fully restores the catalytic utility of the film. The volume phase transition phenomenon can be, therefore, exploited to deliver/preserve the enzyme, purify the gel matrix, or control the catalytic process.
We propose a new system for the immobilization of redox enzymes exemplified by laccase. The strength of the adhesion of the gel layers to ITO was high enough to keep the layer intact during a few months for more than 90% of the samples. The enzyme immobilized in the NIPA gel is in electric contact with electrode, and the electrochemistry of laccase active sites can be seen. Potentials of reduction of T1 and T2 sites of Cerrena unicolor laccase were determined to be 0.79 and 0.38 V, respectively. The enzyme retains its activity in the polymer matrix. The NIPA gel used does not change the affinity of mediators. The temperature-dependent phase transition of the gel gives the possibility to control the catalytic efficiency of the film by changing temperature. Shrinking of the gel considerably decreases the efficiency of the catalytic reaction, however, interestingly, does not lead to any unwanted changes in the enzyme structure. Upon reswelling of the gel at temperatures lower than 30 °C the catalytic efficiency returns to its original value. This property allows purification of the reaction environment. The volume phase transition phenomenon can be exploited to preserve the enzyme or to deliver it to a selected place. The modified electrode can be easily miniaturized and contains no metallic parts. The measurements proved efficient catalytic reduction of oxygen in the gel and the utility of the electrode for oxygen monitoring in solutions. Acknowledgment. This work has been financially supported by the Polish Ministry of Sciences and Higher Education Grant No. N N204 240134 and by the University of Warsaw through Grant BW-179206. References and Notes (1) Solomon, E. I.; Sundaram, U. M. T.; Machonkin, E. Chem. ReV. 1996, 96, 2563–2606. (2) Barton, S. C.; Pickard, M.; Vazquez-Duhalt, R.; Heller, A. Biosens. Bioelectron. 2002, 17, 1071–1074. (3) Barton, S. C.; Kim, H.-H.; Binyamin, G.; Zhang, Y.; Heller, A. J. Am. Chem. Soc. 2001, 123, 5802–5803. (4) Barriere, F.; Ferry, Y.; Rochefort, D.; Leech, D. Electrochem. Commun. 2004, 6, 237–241. (5) Stolarczyk, K.; Nazaruk, E.; Rogalski, J.; Bilewicz, R. Electrochem. Commun. 2007, 9, 115–118. (6) Nazaruk, E.; Michota, A.; Bukowska, J.; Shleev, S.; Gorton, L.; Bilewicz, R. J. Biol. Inorg. Chem. 2007, 12, 335–344. (7) Nazaruk, E.; Bilewicz, R. Bioelectrochemistry 2007, 71, 8–14.
Immobilization of Laccase on ITO Electrodes (8) Stolarczyk, K.; Nazaruk, E.; Rogalski, J.; Bilewicz, R. Electrochim. Acta 2008, 53, 3983–3990. (9) Nazaruk, E.; Bilewicz, R.; Lindblom, G. B.; Lindholm-Sethson, B. Anal. Bioanal. Chem. 2008, 391, 1569. (10) Nazaruk, E.; Smolin˜ski, S.; Swatko-Ossor, M.; Ginalska, G.; Fiedurek, J.; Rogalski, J.; Bilewicz, R. J. Power Sources 2008, 183, 533. (11) Rowinski, P.; Bilewicz, R.; Stebe, M.-J.; Rogalska, E. Anal. Chem. 2004, 76, 283–291. (12) Thurston, Ch. F. Microbiology 1994, 140, 19–26. (13) Mayer, A. M.; Staples, R. C. Phytochemistry 2002, 60, 551–565. (14) Jarosz-Wilkolazka, A.; Janusz, G.; Ruzgas, T.; Gorton, L.; Malarczyk, E.; Leonowicz, A. Anal. Lett. 2004, 37, 1497–1513. (15) Jarosz-Wilkolazka, A.; Ruzgas, T.; Gorton, L. Talanta 2005, 66, 1219–1224. (16) Haghighi, B.; Jarosz-Wilkolazka, A.; Ruzgas, T.; Gorton, L.; Leonowicz, A. Int. J. EnViron. Anal. Chem. 2005, 85, 753–770. (17) Boidin, J. ReV. Mycol. 1951, 16, 173–197. (18) Yoshida, H. J. Chem. Soc. Trans. 1883, 43, 472–486. (19) Givaudan, A.; Effosse, A.; Faure, D.; Potier, P.; Bouillant, M. L.; Bally, R. FEMS Microbiol. Lett. 1993, 108, 205–210. (20) Barrett, F. M.; Andersen, S. O. Insect Biochem. 1981, 11, 17–23. (21) Leonowicz, A.; Gianfreda, L.; Rogalski, J.; Jaszek, L. M.; Luterek, J. M.; Wasilewska, W.; Malarczyk, E.; Dawidowicz, E.; Fink-Boots, A. M.; Ginalska, G.; Cho, N.-S. Palpu, Chongi Gisul 1997, 29, 7–17. (22) Luterek, J.; Gianfreda, L.; Wojtas-Wasilewska, M.; Rogalski, J.; Jaszek, M.; Malarczyk, E.; Dawidowicz, A.; Finksboots, M.; Ginalska, G.; Leonowicz, A. Acta Microbiol. Pol. 1997, 46, 297–311. (23) Ogawa, K.; Nakajima-Kambe, T.; Nakahara, T.; Kokufuta, E. Biomacromolecules 2002, 3, 625–631. (24) Tumturk, H.; Karaca, N.; Demirel, G.; Sahin, F. Int. J. Biol. Macromol. 2007, 40, 281–285. (25) Ogawa, K.; Kokufuta, E. Langmuir 2002, 18, 5661–5667. (26) Janusz, G. Ph.D. Thesis, UMCS Lublin, 2005; p 222. (27) Leonowicz, A.; Grzywnowicz, K. Enzyme Microb. Technol. 1981, 3, 55–58.
J. Phys. Chem. B, Vol. 113, No. 17, 2009 6067 (28) Bradford, M. M. Anal. Biochem. 1976, 72, 248–254. (29) Shleev, S.; Tkac, J.; Christenson, A.; Ruzgas, T.; Yaropolov, A. I.; Whittaker, J. W.; Gorton, L. Biosens. Bioelectron. 2005, 20, 2517–2554. (30) Soukharev, V.; Mano, N.; Heller, A. J. Am. Chem. Soc. 2004, 126, 8368–8369. (31) Barriere, F.; Kavanagh, P.; Leech, D. Electrochim. Acta 2006, 51, 5187–5192. (32) Shleev, S.; Klis, M.; Wang, Y.; Rogalski, J.; Bilewicz, R.; Gorton, L. Electroanalysis 2007, 19, 1039–1047. (33) Shleev, S.; Jarosz-Wilkolazka, A.; Khalunina, A.; Morozova, O.; Yaropolov, A.; Ruzgas, T.; Gorton, L. Bioelectrochemistry 2005, 67, 115– 124. (34) Shleev, S.; Pita, M.; Yaropolov, A. I.; Ruzgas, T.; Gorton, L. Electroanalysis 2006, 18, 1901–1908. (35) Reinhammar, B. R. M. Biochim. Biophys. Acta 1972, 275, 245– 259. (36) Christenson, A.; Shleev, S.; Mano, N.; Heller, A.; Gorton, L. Biochim. Biophys. Acta 2006, 1757, 1634–1641. (37) Kawahara, K.; Suzuki, S.; Sakurai, T.; Nakahara, A. Arch. Biochem. Biophys. 1985, 241, 179–186. (38) Rogalski, J.; Dawidowicz, A. L.; Leonowicz, A. Acta Biotechnol. 1990, 10, 261–269. (39) Zawisza, I.; Rogalski, J.; Opallo, M. J. Electroanal. Chem. 2006, 588, 244–252. (40) Karbarz, M.; Gniadek, M.; Stojek, Z. Electroanalysis 2005, 17, 1397–1400. (41) Shu, F. R.; Wilson, G. S. Anal. Chem. 1976, 48, 1679–1686. (42) Klis, M.; Rogalski, J.; Bilewicz, R. Bioelectrochemistry 2007, 71, 2–7. (43) Hirokawa, Y.; Tanaka, T. J. Chem. Phys. 1984, 81, 6379–6380. (44) Karbarz, M.; Pulka, K.; Misicka, A.; Stojek, Z. Langmuir 2006, 22, 7843–7847.
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