Anal. Chem. 1996, 68, 4335-4341
Improvement of Electrochemical Biosensors Using Enzyme Immobilization from Water-Organic Mixtures with a High Content of Organic Solvent Arkady A. Karyakin,† Elena E. Karyakina,† and Lo Gorton*
Department of Analytical Chemistry , University of Lund, P.O. Box 124, S-221 00 Lund, Sweden Oksana A. Bobrova, Lylia V. Lukachova, Alexander K. Gladilin, and Andrey V. Levashov
Chemical Department, M. V. Lomonosov Moscow State University, 119899 Moscow, Russia
We propose immobilizing enzymes into Nafion membranes by suspending the enzyme in a water-ethanol mixture with a high (>90%) ethanol content, followed by mixing with the dissolved polyelectrolyte, and finally allowing the enzyme-polyelectrolyte solution to dry at the target surface (electrode surface). Since Nafion solution was deposited from a solution where it is truly dissolved and without excessive dilution with water, the resulting membranes were more uniform and stable than those otherwise obtained. Enzyme suspensions in concentrated ethanol solutions were prepared without any prior modifications of the protein. The remaining activity after 30 min exposure to such solutions under optimal conditions was up to 100%. The stability of the enzymes in these suspensions was higher than that in aqueous solution. Electrochemical biosensors made accordingly showed a several times increased response compared with those of enzyme electrodes based on the traditional way of using excessive dilution of Nafion with water. The remaining activity, after the drying-washing cycle of the enzyme electrode made by enzyme immobilization from concentrated organic solvent, was at least 10 times higher than that of the traditional one. Operation of electrochemical biosensors requires a conjugation of the biochemical and electrochemical reactions. For this aim, the biological recognition element should be immobilized at the electrode surface. There are two principal ways of achieving such immobilization: covalent linking and the entrapment into gel or polymer matrices. However, the former method is not suitable for enzyme immobilization onto the surface of modified electrodes, where the modifier is used as an electrocatalyst of an oxidationreduction reaction of the conjugated enzyme substrates or products. To link a sufficient amount of enzyme, it is necessary to treat the modified electrode with an excess of a bifunctional reagent. That may cause an inactivation of the electrocatalyst. Moreover, sometimes the modified electrode surface contains no functional groups, as in the case of redox-active inorganic polycrystals.1 † Permanent address: Chemical Department, M. V. Lomonosov Moscow State University. (1) Karyakin, A. A.; Gitelmacher, O. V.; Karyakina, E. E. Anal. Chem. 1995, 67, 2419-2423.
S0003-2700(96)00397-6 CCC: $12.00
© 1996 American Chemical Society
In the search for suitable matrices for enzyme immobilization, there has been a growing interest in the entrapment/binding of the enzyme into the polyelectrolyte Nafion. This polymer is commercially available as a solution in a mixture of 90% low-chain aliphatic alcohols and 10% water. The method of membrane formation is a simple dipping of the electrode into the polyelectrolyte solution or syringing a small volume of the solution onto the electrode surface and allowing the solvent to evaporate. The resulting membranes possess a high adhesion to the surface and a low swelling in aqueous media. In addition, the polyelectrolyte membrane stabilizes the ionic strength at the electrode surface essential for sensor applications. About 15 years ago, the entrapment of positively charged redox-active compounds into Nafion membranes was reported.2,3 The high operation stability of the resulting redox active films allowed authors to propose them for a variety of electrochemical applications, even as reference electrodes.4 Nafion has already found a wide use in the field of biosensor development. Among its main advantages is that Nafion provides a biocompatible interface with mammalian tissue and hence offers the potential for use with implantable sensors.5 Nafion encapsulation also prevents electrode fouling and increases the dynamic range of analyte detection.6-8 Being a negatively charged polyelectrolyte matrix, Nafion reduces the permeability to negatively charged substances. Hence, using an additional Nafion membrane, one can improve the sensor selectivity9-11 and practically eliminate the influence of reductants such as ascorbate10,12 and acetaminophen13 on the biosensor response. Another special application of Nafion films with regard to biosensor development is the immobilization of mediators used (2) Rubinstein, I.; Bard, A. J. J. Am. Chem. Soc. 1980, 102, 6641-6642. (3) Rubinstein, I.; Bard, A. J. J. Am. Chem. Soc. 1981, 103, 5007-5013. (4) Rubinstein, I. Anal. Chem. 1984, 56, 1135-1137. (5) Turner, R. F. B.; Harrison, D. J.; Rajotte, R. V.; Baltes, H. P. Sens. Actuators 1990, B1, 561-564. (6) Chen, C. Y.; Tamiya, E.; Ishihara, K.; Kosugi, Y.; Su, Y. C.; Nakabayashi, N.; Karube, I. Appl. Biochem. Biotechnol. 1992, 36, 211-226. (7) Chen, C. Y.; Gotoh, M.; Makino, H.; Su, Y. C.; Tamiya, E.; Karube, I. Anal. Chim. Acta 1992, 265, 5-14. (8) Ohara, T. J.; Rajagopalan, R.; Heller, A. Anal. Chem. 1994, 66, 2451-2457. (9) Wightman, R. M. In Biosensor Technology; Buck, R. P., Hatfield, W. E., Umana, M., Bowden, E. F., Eds.; Marcel Dekker: New York, 1990; pp 3954. (10) Navera, E. N.; Suzuki, M.; Tamiya, E.; Takeuchi, T.; Karube, I. Electroanalysis 1993, 5, 17-22. (11) Garguilo, M. G.; Michael, A. C. Anal. Chim. Acta 1995, 307, 291-299.
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in bioelectrocatalytic reactions. Based on the mentioned high operational stability of the entrapped positively charged redoxactive compounds,2,3 one can create a mediator-containing layer between the electrode and the immobilized enzyme. Indeed, several amperometric glucose electrodes were developed using this approach. To transfer electrons from the active site of glucose oxidase (GOx) to the electrode, different mediators were used, e.g., ferrocene14 and tetrathiofulvalene.15 Nafion membranes have already been used for immobilizing GOx16 and glucose, alcohol, malate, and L-lactate dehydrogenase.17 The method included syringing an appropriate amount of deposition solution and allowing the solvent to evaporate. Nafionenzyme films were made by sequential deposition of the enzyme solution and the solution of the polyelectrolyte17 or by deposition of a mixture of the enzyme and the Nafion solutions.16,18 To protect the enzyme against inactivation by the organic solvent, the initial Nafion solution was diluted with water or with an aqueous buffer. As a result, the Nafion membranes were formed from an emulsion of the polymer instead of the “real” solution (in the sense that Nafion is truly dissolved as a molecular solution as opposed to a colloid dispersion or an emulsion). Through deposition of the polymer from its suspension or emulsion, one obviously cannot obtain uniform membranes. Investigations of the permeability of Nafion films made from an alcohol solution and from water-alcohol mixtures with a low alcohol content were reported.16 A similar penetration barrier to anodic species was achieved only when the membrane formed from an water-alcohol mixture was 10 times thicker than that formed from an undiluted preparation. Because the polyelectrolyte membranes are not uniform, low rates of charge transfer were obtained between the enzyme and the electrode in a NafionGOx-ferrocene layer.19 Our previous experience with Nafion membranes formed from water-alcohol mixtures with a low alcohol content showed that they were also not sufficiently stable. To improve the structure and stability of the enzyme-containing Nafion membranes, one should perform the deposition from a solution where the polyelectrolyte is truly dissolved. It is possible to dissolve enzymes in organic solvents using reversed micelles.20 The entrapment of GOx into liposomes prior to immobilization in the Nafion film has already been done. However, it was curious that the authors deposited the Nafion layer not from a true solution but when excessively diluted with water.21,22 The properties of enzymes in organic solvents and waterorganic mixtures have been intensively studied. One of the main reasons for such an interest was an increase in the solubility of (12) Matuszewski, W.; Trojanowicz, M.; Lewenstam, A. Electroanalysis 1990, 2, 607-615. (13) Zhang, Y.; Hu, Y.; Wilson, G. S.; Moatti-Sirat, D.; Poitout, V.; Reach, G. Anal. Chem. 1994, 66, 1183-1188. (14) Dong, S.; Wang, B.; Liu, B. Biosens. Bioelectron. 1992, 7, 215-222. (15) Liu, H.; Deng, J. Electrochim. Acta 1995, 40, 1845-1849. (16) Fortier, G.; Vaillancourt, M.; Belanger, D. Electroanalysis 1992, 4, 275283. (17) Karyakin, A. A.; Karyakina, E. E.; Schuhmann, W.; Schmidt, H.-L.; Varfolomeyev, S. D. Electroanalysis 1994, 6, 821-829. (18) Rishpon, J.; Gottesfeld, S.; Campbell, C.; Davey, J.; Zawodzinski, T. A. J. Electroanalysis 1994, 6, 17-21. (19) Harkness, J. K.; Murphy, O. J.; Hitchens, G. D. J. Electroanal. Chem. 1993, 357, 261-272. (20) Martinek, K.; Berezin, I. V.; Khmelnitsky, Y. L.; Klyachko, N. L.; Levashov, A. V. Biocatalysis 1987, 1, 9-15. (21) Mizutani, F.; Yabuki, S.; Katsura, T. Anal. Chim. Acta 1993, 274, 201207. (22) Mizutani, F.; Yabuki, S. Biosens. Bioelectron. 1994, 9, 411-414.
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hydrophobic compounds and thus a shift of the reaction equilibrium in favor of the desired products.23,24 The dependence of the enzyme activity on the concentration of organic cosolvent in water-organic mixtures usually consists of three typical regions. The first one (region I) corresponds to a low content of organic solvent, where there is no significant decrease in enzyme activity. When increasing the concentration of the organic solvent, a critical (“threshold”) value25 will be reached. At concentrations higher than the threshold value, region II is found, where the enzyme undergoes conformational changes and completely loses its catalytic activity. However, already at the beginning of the century, the existence of a third region (III) was indicated.26 In concentrated organic solutions with a water content less than 1015%, the enzyme activity reappears again. The reason is that, in such media, the enzyme becomes insoluble and occurs in a suspension. The enzyme molecules situated inside the colloid particle are preserved against inactivation by the interfacial molecules. In this region III, the enzymes are less active than in aqueous solutions but are rather stable.27-29 Because such suspensions have high stability, several methods of preparative enzyme precipitation from water solutions using water miscible organic solvents are known.30 Based on the discussion above, we believed that it would be possible to immobilize enzymes without prior modifications from water-organic mixtures with a high content of organic solvent. An improvement in the stability of the Nafion-enzyme membranes was expected, because Nafion is soluble in such mixtures. An important question in this case concerns the compatibility of the enzymes with polyelectrolytes. However, it was found that polyelectrolytes stabilized enzymes in water-organic mixtures, increasing the threshold concentration.31 The aim of the present paper is to investigate the immobilization of GOx and alcohol dehydrogenase (ADH) in Nafion membranes from water-ethanol solutions with a high ethanol content. The oxidase- and dehydrogenase-based biosensors were developed to exhibit the advantages of the immobilization method proposed. EXPERIMENTAL SECTION Materials. All experiments were carried out using pure water. It was prepared by gentle distillation of ordinary distilled water with potassium permanganate in acidic medium. All inorganic salts and glucose were obtained at the highest purity and afterward were recrystallized from pure water. Acetaldehyde was distilled prior to use. Absolute ethanol was prepared by distillation of sodium alcoholate and used immediately. Nafion (5% solution in 90% low-chain aliphatic alcohols) and Methylene Green were obtained from Aldrich (Steinheim, Germany). NADH and NAD+ were obtained from Sigma (St. Louis, MO). Yeast alcohol dehydrogenase (ADH, EC 1.1.1.1, Reanal, Budapest, Hungary) (23) Khmelnitsky, Y. L.; Levashov, A. V.; Klyachko, N. L.; Martinek, K. Enzyme Microb. Technol. 1988, 10, 710-724. (24) Gupta, M. N. Eur. J. Biochem. 1992, 203, 25-32. (25) Khmelnitsky, Y. L.; Mozhaev, V. V.; Belova, A. B.; Sergeeva, M. V.; Martinek, K. Eur. J. Biochem. 1991, 198, 31-41. (26) Bourquelot, E.; Bridel, M. Ann. Chim. Phys. 1913, 29, 145-218. (27) Zaks, A.; Klibanov, A. M. J. Biol. Chem. 1988, 263, 3194-3201. (28) Kise, H.; Shirato, H. Enzyme Microb. Technol. 1988, 10, 582-585. (29) Tomiuchi, Y.; Ohshima, K.; Kise, H. Bull. Chem. Soc. Jpn. 1992, 65, 25992603. (30) Dixon, M.; Webb, E. C. Enzymes; Longmans: Oxford, 1964. (31) Gladilin, A. K.; Kudryashova, E. V.; Vakurov, A. V.; Izumrudov, V. A.; Mozhaev, V. V.; Levashov, A. V. Biotechnol. Lett. 1995, 17, 1329-1334.
had an initial activity of 10 IU/mg. Glucose oxidase (GOx, EC 1.1.3.4) from Aspergillus niger (type VII-S, 180 IU/mg) was produced by Sigma. Glucose oxidase from Penicillium vitale was generously obtained as a gift from Dr. Yu. Rodionov (Moscow State University, Russia) and had an initial activity of 100 IU/mg. All other chemicals used were of analytical grade. Instrumentation. Electrochemical measurements were performed using a Solartron Schlumberger Model 1286 electrochemical interface (Farnborough, UK), a Universal polarograph OH 105 (Budapest, Hungary), and an electrochemical analyzer from Bioanalytical Systems (BAS-100W, West Lafayette, IN). The activity of ADH was measured using an LKB Ultraspec U-II spectrophotometer (Bromma, Sweden). Fitting of experimental data and preparation of the manuscript were done using an IBMcompatible 486 Jetbook computer. Electrochemical Methods. A three-compartment electrochemical cell was used containing a platinum net auxiliary electrode and an Ag/AgCl reference electrode in 1 M KCl. The cell construction allowed deaeration of the working electrode space. Glassy carbon disk electrodes (diameter 1.5 mm) were used as working electrodes. Prior to use, the glassy carbon electrodes were mechanically polished with aluminum powder (Al2O3, 5 µm, Chemapol, Czech Republic) until a mirror finish was observed and further treated electrochemically in 0.2 M sulfuric acid according to the following procedure: first the electrode was held for 3 min at 1.6 V, followed by cycling between -0.5 and 1.5 V at a sweep rate of 40 mV/s for approximately 10 min. Afterward a stable voltammogram of the glassy carbon electrode was obtained. Preparations of Modified Electrodes. The electropolymerization of Methylene Green was carried out by cycling the applied potential in the range between -0.3 (start potential) and 1.3 V at a sweep rate of 50 mV/s as was previously described.32 The “growing solution” consisted of 0.4 mM Methylene Green in 10 mM sodium tetraborate (pH 9.1), also containing 0.1 M NaNO3. The growing conditions were chosen in accordance with the influence of counterion and solution pH on the electropolymerization of Methylene Blue.33 The electrochemically active electrode coverage with electropolymerized Methylene Green was between 5 and 7 nmol/cm2, evaluated with cyclic voltammetry. Electrochemical synthesis of self-doped polyaniline was done similarly to a method previously described.34 The initial solution used for electropolymerization contained 50 mM aniline and 50 mM m-aminobenzenesulfonic acid in 0.5 M H2SO4. Films were grown by sweeping the applied potential from -0.1 up to 0.8 V, keeping at the anodic switching potential during 5 s and sweeping back to the initial potential. The electropolymerization occurred at a sweep rate of 50 mV/s, within approximately 30-40 cycles. After deposition, the polyaniline films were discharged in 1 mM HCl at a potential of 0.5 V to remove side products of the electrooxidation of aniline. The total charge passed after recharging of the deposited polymer was ∼10 mC/cm2. Electrodeposition of Prussian Blue was done by applying a constant potential of 0.4 V for 30 s when the electrode was dipped into a solution initially containing 1 mM K3[Fe(CN)6] and 1 mM (32) Karyakin, A. A.; Karyakina, E. E.; Schlereth, D. D.; Gleixner, G.; Schuhmann, W.; Schmidt, H.-L. J. Electroanal. Chem., submitted. (33) Karyakin, A. A.; Strakhova, A. K.; Karyakina, E. E.; Varfolomeyev, S. D.; Yatsimirsky, A. K. Bioelectrochem. Bioenerg. 1993, 32, 35-43. (34) Karyakina, E. E.; Neftyakova, L. V.; Karyakin, A. A. Anal. Lett. 1994, 27, 2871-2882.
FeCl3. The supporting electrolyte was 1 M KCl, also containing 3 mM HCl. After deposition, the Prussian Blue films were activated in 0.1 M KCl by cycling the applied potential between -0.05 and 0.35 V at a sweep rate of 50 mV/s. The total amount of deposited Prussian Blue was between 2 and 3 nmol/cm2, assuming the transfer of four electrons per unit cell.35 Preparation of Enzyme Membranes. The enzyme suspension in a water-ethanol mixture with a high (>85%) ethanol content was prepared as follows: the lyophilized enzyme samples were dissolved in water to final concentrations of ADH of 80100 mg/mL and of GOx of 20-30 mg/mL. It was shown that such concentrations caused no enzyme inactivation. Finally, the enzyme suspension was made by addition of pure ethanol or a water-ethanol mixture to the enzyme solution. All percentage units related to the water-alcohol mixtures are volume percents. The enzyme suspensions were prepared in water-ethanol mixtures with a water content in the range of 2.5-15%. The suspensions were kept for 30 min at room temperature. Then, small samples of the suspensions were injected into substrate solutions in a spectrophotometric cuvette or a Clark electrode cell for ADH and GOx, respectively. The final ethanol content in the substrate solutions was
