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Novel Conducting Polymer Electrolyte Biosensor Based on Poly(1-vinyl imidazole) and Poly(acrylic acid) Networks Ahu Arslan,† Senem Kıralp,† Levent Toppare,*,† and Ayhan Bozkurt‡ Department of Chemistry, Middle East Technical UniVersity, 06531 Ankara, Turkey and Department of Chemistry, Fatih UniVersity, 34900 Buyukcekmece, Istanbul, Turkey ReceiVed NoVember 12, 2005. In Final Form: January 23, 2006 Biosensor construction and characterization studies of poly(acrylic acid) (PAA) and poly(1-vinyl imidazole) (PVI) complex systems have been carried out. The biosensors were prepared by mixing PAA with PVI at several stoichiometric ratios, x (molar ratio of the monomer repeat units). The enzyme, invertase, was entrapped in the PAA/PVA interpenetrating polymer networks during complexation. Modifications were made on the PAA/PVI conducting polymer electrolyte matrixes to improve the stability and performance of the polymer electrolyte-based enzyme biosensor. The maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were investigated for the immobilized invertase. The temperature and pH optimization, operational stability, and shelf life of the polymer electrolyte biosensor were also examined.
Introduction The discovery of polymer-salt complexes as well as the recognition of their potential application as solid electrolytes resulted in the development of numerous polymer electrolytes, including those with a protonic type of conductivity.1-3 Entirely different classes of materials have attracted increasing attention as proton conductors including polymers, oxide ceramics, and intercalation compounds.4-7 It is well established that proton-conducting polymer electrolytes can be obtained by doping polymers bearing ether, alcohol, imine, amide, or imide groups with acid.8-12 This very simple concept is now extended to more complex systems where either an inorganic filler or a plasticizer or both are added to the binary polymer blend to improve the properties of the polymer matrix. Hydrophilic polymers such as poly(vinyl alcohol) and poly(vinyl imidazole) can be impregnated with an acid and behave as solid proton conductors. The neutralization of polymeric bases may be a more effective way to generate various proton sources within a polymer matrix. In comparison with the usual liquid electrolytes, the polymer/ acid systems have obvious advantages in terms of reduced leakage and corrosion problems, and they represent less costly alternatives to perfluorinated polymers. The main advantage compared with * Corresponding author. E-mail:
[email protected]. Tel: +90-3122103251. Fax: +90-312-2101280. † Middle East Technical University. ‡ Fatih University. (1) Donoso, P.; Gorecki, W.; Berthier, C.; Defendini, P.; Poinsignon, C.; Armand, M. Solid State Ionics 1988, 28-30, 969-974. (2) Petty-Weeks, S.; Zupancic, J. J.; Swedo, J. R. Solid State Ionics 1988, 31, 117-125. (3) Przyluski, J.; Wieezorek, W. Synth. Met. 1991, 45, 323-333. (4) Kreuer, K. D. Solid State Ionics 1997, 97, 1-15. (5) Flint, S. D.; Slade, R. C. T. Solid State Ionics 1997, 97, 299-307. (6) Baranov, A. I.; Sinitsyn, V. V.; Vinnichenko, V. Y.; Jones, D. J.; Bonnet, B. Solid State Ionics 1997, 97, 153-160. (7) Murugaraj, P.; Kreuer, K. D.; He, T.; Schober, T.; Maier, J. Solid State Ionics 1997, 98, 1-6. (8) Lasse´gues, J. C.; Desbat, B.; Trinquet, O.; Cruege, F.; Poinsignon, C. Solid State Ionics 1989, 35, 17-25. (9) Grondin, J.; Rodriguez, D.; Lasse´gues, J. C. Solid State Ionics 1995, 77, 70-75. (10) Polak, A.; Petty-Weaks, S.; Buehler, A. J. Sens. Actuators 1986, 9, 1-7. (11) Kawahara, M.; Morita, J.; Rikukawa, M.; Sanui, K.; Ogata, N. Electrochim. Acta 2000, 45, 1395-1398. (12) Bozkurt, A.; Ise, M.; Kreuer, K. D.; Meyer, W. H.; Wegner, G. Solid State Ionics 1999, 125, 225-233.
Figure 1. Protonation of PVI via doping with PAA.
conventional proton-conducting polymers is that it is relatively inexpensive. Polymer electrolytes are under extensive investigation for applications in fuel cells, hydrogen sensors, and electrochromic devices.13,14 Proton conductors can be classified according to the preparation method, chemical composition, structural dimensionality, mechanism of conduction, and so forth. Here, a proton-conducting material is discussed according to the range of temperature in which it can be used in technological applications. Attempts to develop proton-conducting materials suitable for medium temperature are being made in many laboratories.15,16 Recently, proton-conducting polymer electrolytes based on polymer heterocycle hybrid electrolytes show improved thermal properties, and they are suitable for biosensor applications. The proton conductivity and stability of these polymer matrixes show an important dependence on temperature as x (molar ratio of the monomer repeat units) is varied. The formation of proton defects is necessary for proton conduction. Heterocycles such as imidazoles have been reported in this respect.17 Their nitrogen sites act as strong proton acceptors that form protonic charge carriers. The protonation of PVI via doping with PAA is assumed in this direction (Figure 1). (13) Colomban, P., Ed.; Proton Conductors; Solids, Membranes and Gelss Materials and DeVices; Cambridge University Press: Cambridge, U.K., 1992. (14) Wainright, J. S.; Wang, J. T.; Weng, D.; Savinell, R. F.; Litt, M. H. J. Electrochem. Soc. 1995, 142, L121-L123. (15) Alberti, G.; Costantino, U.; Casciola, M.; Ferroni, S.; Massinelli, L.; Staiti, P. Solid State Ionics 2001, 145, 249-255. (16) Bozkurt, A.; Meyer, W. H.; Wegner, G. J. Power Sources 2003, 123, 126-131. (17) Kreuer, K. D.; Fuchs, A.; Ise, M.; Spaeth, M.; Maier, J. Electrochim. Acta 1998, 43, 1281-1288.
10.1021/la0530539 CCC: $33.50 © 2006 American Chemical Society Published on Web 02/16/2006
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Figure 2. FTIR results for PAA and PAA/PVI polymer electrolytes: (a) pure PAA, (b) PAA/0.25PVI, (c) PAA/0.5PVI, and (d) PAA/1.0PVI.
β-Fructofuranosidase (E.C. no. 3.2.1.26) catalyzes the hydrolysis of sucrose to glucose and fructose, which is known as the invert sugar. Sucrose crystallizes more readily than invert sugar, so the latter is widely used in the production of noncrystallizing creams, in making jam and artificial honey. Invertase occurs in the small intestine of mammals and in the tissues of certain animals and plants. It may be obtained in a relatively pure state from yeast, which is a very good source. Although, invertase has a lower probability of achieving commercial use in its immobilized form, it is one of the most studied of all enzymes because it is a model enzyme for experimental purposes.18-20 This article reports on some results of studies carried out with a new conducting polymer electrolyte biosensor (PVI/PAA). The main goal of this work is to prepare a biosensor via immobilizing invertase into a polymer electrolyte matrix. We report the synthesis and properties of the PAA/PVI polymer electrolyte biosensor. We expect that the polymer electrolyte matrix will be able to improve the properties of the enzyme biosensor, and we propose that the polymer electrolyte matrix will show excellent temperature properties that are very important in preventing the denaturation of enzymes at high temperatures. Method Materials. Invertase (E.C. no. 3.2.1.26) was purchased from Sigma. PVI was synthesized by solution polymerization,21 and PAA (Mn ) 1000) was synthesized by conventional radical polymerization of acrylic acid (Merck).16 Sucrose was obtained from Sigma. For the spectrophotometric activity determination, the Nelson method was used. For acetate buffer, acetic acid and sodium acetate were used. A Shimadzu UV-1601 model spectrophotometer and a JEOL JSM-6400 model scanning electron microscope (SEM) were used. The IR spectra of the samples were obtained with a Nicolet 510 FT-IR spectrophotometer. Entrapment of Invertase in a Polymer Electrolyte Matrix. PVI and PAA were dissolved with various stoichiometric ratios in an enzyme solution, and solutions were stirred to obtain the polymer electrolyte. PAA (0.0152 g) and PVI (0.02 g) (1:1 ratio of repeating (18) Kizilyar, N.; Akbulut, U.; Toppare, L.; O ¨ zden, M.; Yagci, Y. Synth. Met. 1999, 104, 45-50. (19) Erginer, R.; Toppare, L.; Alkan, S.; Bakir, U. React. Funct. Polym. 2000, 45, 227-233. (20) Kiralp, S.; Toppare, L.; Yapci, Y. Synth. Met. 2003, 135, 79-80. (21) Li, X.; Goh, S. H.; Lai, Y. H.; Wee, A. T. S. Polymer 2001, 42, 54635469.
units of the polymers) have a maximum capacity of absorbing 0.4 mL of water. The enzyme solution was prepared in pH 5.1 acetate buffer with an enzyme concentration of 1.0 mg/mL. Finally, polymer-enzyme solutions were stirred to obtain the polymer electrolyte biosensor. All solutions were completely converted to the polymer electrolyte matrix (100% conversion was obtained). As a result, the whole enzyme was incorporated into the matrix. This matrix was used in activity determinations after preparation. The immobilization of invertase was achieved via physical entrapment during the complexation process. Determination of Invertase Activity. The activity determination was performed by using Nelson’s method.22 Different concentrations of sucrose solutions prepared in acetate buffer (pH 5.1) were kept in a water bath at 25 °C for 5 min. The enzyme biosensor was immersed in the test tubes and shaken for different incubation times(2, 4, and 6 min). Then, the biosensor was taken out of the solution, and 1.0 mL of Nelson’s reagent was added to 1.0 mL of the substrate solution to stop the enzymatic reaction. The test tubes were kept in boiling water for 20 min to terminate the enzymatic reaction completely, and then they were cooled to room temperature. Finally, 1.0 mL of an arsenomolybdate solution and 7.0 mL of distilled water were added to obtain a colored complex. After mixing, absorbances were determined at 540 nm. Determination of Kinetic Parameters. To determine the maximum reaction rate (Vm) and the Michaelis-Menten constant (Km), the activity assay was applied for different substrate (sucrose) concentrations. Sucrose solutions (0.05/0.1/0.2/0.4/0.6/1.0/1.5 mol/L) were prepared in acetate buffer (pH 5.1) and kept in a water bath at 25 °C for 5 min, and then the enzyme biosensor or free enzyme solution was added to the test tubes and shaken for incubation times of 2, 4, and 6 min. Both the free and immobilized enzyme concentrations were 1.0 mg/mL. Determination of Optimum Temperature and pH. Optimum temperatures were determined by changing temperature between 10 and 80 °C while keeping the substrate concentration constant (10Km). In addition to the optimum temperature determination, improvement of the biosensor was achieved depending on the polymer composition. The PAA/PVI polymer electrolyte was synthesized in different stoichiometric ratios (x ) [moles of VI in PVI]/[mole of AA in PAA]). In the present work, temperature optimization was done for x ) 1.0, 0.5, and 0.25. Also, pH optimizations were carried out by changing the pH range between 2 and 11 at constant temperature (25 °C). Operational Stabilities and Shelf Life. The operational stability of the enzyme biosensor was determined at optimum activity (22) Nelson, N. J. Biol. Chem. 1944, 153, 375-380.
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Table 1. Percent Protonation of PVI with PAA in Different Stoichiometric Ratios x (mol ratio of the monomer repeat units, m/n)
% protonation
0.25 0.5 1.0
90 91 94
PAA-P(VI)0.25 PAA-P(VI)0.5 PAA-P(VI)1.0
Table 2. Kinetic Parameters for Free and Immobilized Invertase
free invertase PAA/PVI/invertase
Km (mM)
Vmax (µmol/min mL)
24.3 112.6
82.3 5.8
Figure 3. Optimum temperature of the enzyme biosensor.
conditions by using electrodes in 30 activity assays per day. The shelf life of the electrodes was investigated by performing activity measurements within 25 days.
Results and Discussion FTIR Results. FTIR results of the PAA and PAA/PVI polymer electrolytes are given in Figure 2. A strong absorption was observed at 1716 cm-1 (CdO stretching), 1171, and 1249 cm-1 representing the C-O-H stretching of carboxylic acid groups of the PAA. After polymer electrolyte formation, a new peak was observed at 1547 cm-1, and the intensity of the carbonyl peak at 1716 cm-1 decreased because of the protonation of PVI with PAA. The percent protonation of the polymer electrolyte samples was obtained from the considered peak area.16 First, the ratio of the 1716 to 1249 cm-1 peak was calculated for each spectrum. Then, the degree of protonation for each polymer represented in Figure 2b-d was determined by again taking the ratio of these numbers with respect to the ratio found from Figure 2a. Table 1 shows the percent protonation of PVI with PAA in different stoichiometric ratios. On the basis of the increasing imidazole rings, N-H stretching was observed at 3128 cm-1. Kinetic Parameters. Kinetic parameters Km and Vmax for the enzyme biosensor were found from the Lineweaver-Burk plots23 at constant temperature and pH while varying the substrate concentrations. The calculated parameters are given in Table 2. The results show that there is an increase in Km values when compared with that of the free enzyme.24 The increase in the Km value is due to diffusional difficulties. In the presence of the polymer electrolyte, enzyme and substrate interaction and complex formation become more difficult depending on the porosity of the matrix. The compact structure of the matrix causes difficulty for the diffusion of the substrate inside the matrix. The decrease in the Vmax value when compared with the free enzyme reflects this interaction. The PAA/PVI matrix exhibited a higher Km value and also lower activity. This means that the enzyme and substrate do not prefer to come together for a long time in this matrix. Temperature Optimization of the Enzyme Biosensor. The effect of the reaction temperature on the activity is shown in Figure 3. Free invertase completely lost its activity at 50 °C;21 however, the PAA/PVI/invertase sensor for x ) 1.0 showed maxima at 60 °C, and the PAA/PVI/invertase sensor for x ) 0.5 showed maxima at 60 °C and lost only 25% of its activity at 70 °C. In addition, the PAA/PVI/invertase sensor for x ) 0.25 showed improvement toward high temperature, and it has maxima at 70 °C and lost only 20% of its activity at 80 °C. In the presence of (23) Lineweaver, H.; Burk, D. J. Am. Chem. Soc. 1934, 56, 658-666. (24) Alkan, S.; Toppare, L.; Yagci, Y.; Hepuzer, Y. J. Biomat. Sci. Polym. Ed. 1999, 10, 1223-1230.
Figure 4. Optimum pH of the enzyme biosensor.
Figure 5. Operational stability of the enzyme biosensor.
Figure 6. Shelf life of the enzyme biosensor.
smaller amounts of imidazole, enzyme biosensors have higher activity and resistance to high temperature. As mentioned before, this matrix has good resistance to temperature when used in different stoichiometric ratios.16 It is known from the polyelectrolyte chemistry that the preparation of stoichiometric polysalts from a polyacid and a polybase gives rise to insoluble, rigid, thermally stable materials. The temperature dependence is useful in enzyme immobilization and biosensor construction. It is very important to prevent enzyme denaturation
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Figure 7. SEM micrographs of proton-conducting polymer electrolyte matrixes: (a) PAA/PVI and (b) PAA/PVI/invertase.
at high temperatures in application stages. Clearly, this matrix provides a suitable immobilization medium for this enzyme. pH Optimization of the Enzyme Biosensor. Optimum pH values are often important in enzyme assays. pH changes in the medium, especially high and low pH values, cause denaturation. The variation of invertase activity for a pH range from 2 to 11 was investigated for the PAA/PVI matrix. In pH optimization experiments, the stoichiometric ratio of the polymer electrolyte did not cause a significant change in the enzyme activity, as was the case for temperature optimization. Thus, the PAA/PVI matrix was prepared with an x ) 1.0 ratio. The results are presented in Figure 4. The maximum activity of free invertase was observed at pH 5, and immobilized invertase in the PAA/PVI polymer electrolyte matrix showed its maximum activity at pH 10. There is an increase in the enzyme activity from pH 4 to 10. However, in strongly acidic or strongly basic media such as at pH 2 or 11, the polymer electrolyte loses its conformation and degrades to PAA and PVI. As a result of this change in the matrix structure, the enzyme enters the solution and loses its activity because of denaturation in the acidic or basic medium. The optimum pH was shifted toward the alkaline side when compared with the free enzyme. Operational Stability and Shelf Life of the Enzyme Biosensor. The operational stability was obtained by running 30 measurements in the same day (Figure 5). An activity loss of 20% was observed with the fifth use and remained constant for another 25 measurements. Therefore, this matrix exhibited higher stability during 30 measurements. The activity of the enzyme biosensor was measured every 5 days for 25 days (Figure 6). Invertase entrapped in the PAA/PVI matrix lost 50% of its activity in the first 10 days and completely lost its activity within the next 15 days. The rapid, sharp decrease
in enzyme activity might be due to diffusional effects and conformational changes in the enzyme during the storage period. Both the operational stability and shelf life of enzyme biosensors were measured for PAA/PVI matrixes with x ) 1.0. Surface Morphologies of Polymer Electrolyte Matrixes. Surface morphologies of matrixes were analyzed using a JEOL JSM-6400 scanning electron microscope. SEM micrographs of the PAA/PVI matrix and the PAA/PVI/invertase matrix are shown in Figure 7. The surface morphology of the PAA/PVI polymer electrolyte matrix was simple and very smooth. The morphology of enzymeimmobilized films was significantly changed. It was observed that the enzyme immobilization had a strong effect on the film morphology. The smooth structure was damaged by enzyme entrapment because the entrapped molecule is a huge structure so a change in the conformation of the chains is an expected phenomena.
Conclusions In the present work, the entrapment of invertase was successfully achieved in a PAA/PVI polymer electrolyte matrix. This novel approach was found to provide a suitable medium for the immobilization of invertase. The PAA/PVI matrix has very high temperature resistance depending on the amount of PVI in the matrix. Also, the invertase-entrapped matrix revealed reasonable values for pH optimization, operational stability, and shelf life. Entrapped invertase exhibits high stability over a broad pH range due to the protection of invertase by the polymer electrolyte matrix. Operational stability and shelf life results are suitable for biosensor applications. LA0530539
