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A Biochemo-Mechanical System Consisting of Polyampholyte Gels with Coimmobilized Glucose Oxidase and Urease Yukiko Ogawa, Kazuyoshi Ogawa, Benlian Wang, and Etsuo Kokufuta* Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8565, Japan Received November 14, 2000. In Final Form: February 13, 2001 An attempt was made to prepare a biochemo-mechanical system that is capable of converting biochemical energy created as a result of an enzyme reaction into mechanical work through the swelling and shrinking of a gel. The preparation was performed via the physical entrapment of glucose oxidase (GOD) plus urease within an amphoteric polymer network. We used N-isopropylacrylamide (NIPA) as a main constituent of the network into which acrylic acid (AAc) as an anionic monomer and 1-vinylimidazole (VI) as an cationic monomer were introduced via radical polymerization. The gelation was carried out in the presence and the absence of the enzyme(s) by cross-linking with N,N′-methylenebisacrylamide of the monomer mixtures with molar ratios of NIPA/AAc/VI ) 14:1:1 and 14:1:2. The effects of pH and temperature on the swelling degree were studied in 50 mM NaCl solution. The ampholyte gels exhibited an isoelectric point (pI) at pH 5-6 at which the swelling degree reached a minimum value. Therefore, two pH regions bringing about gel swelling lie on either side of pI, that is, pH < 5 and pH > 6. This characteristic allowed the gel to undergo a shrinking-swelling oscillation passing through pI while an increase and a decrease in pH took place via the immobilized enzyme reactions within the gel phase. When the hydrolysis of urea with the immobilized urease raises the pH in the gel, it continues shrinking at pH < pI but swelling at pH > pI after attainment of an extremely collapsed state at pI. Such volume changes take place during a decrease in the pH because of the oxidation of glucose with the immobilized GOD. As a result, the shrinking-swelling oscillation was observed by alternative addition of the two substrates into the outer medium surrounding the gel.
Introduction Since the original use by Bernfeld and Wan1 of an acrylamide gel for the immobilization of enzymes through physical entrapment, a number of articles have reported the preparation, characterization, and application of gelentrapped enzymes. The technology for entrapping enzymes within gels is believed to be well established already. In addition, the nature of gel-entrapped enzymes can now be better understood. Our present interest lies in the development of new concepts in the research field of gelentrapped enzymes. Almost all living systems convert biochemical energy into mechanical work; a good example of this is muscle contraction. It would be interesting to develop gels with immobilized enzymes that undergo changes in shape because of contractile or expansive forces resulting from enzyme reactions. Such immobilized enzymes would be regarded as “biochemo-mechanical systems”2-5 capable of converting biochemical energy created as a result of an enzyme reaction into mechanical work through the swelling and shrinking of the gel, thereby making this type of immobilized enzyme distinct from the more usual sort from the perspective of its utilization as a biocatalyst in a chemical conversion. The study of the construction of biochemo-mechanical systems should begin with the biochemical regulation of * To whom correspondence should be addressed. (1) Bernfeld, P.; Wan, J. Science 1963, 142, 678. (2) Kokufuta, E. Prog. Polym. Sci. 1992, 16, 647. (3) Kokufuta, E. Adv. Polym. Sci. 1993, 110, 159. (4) Kokufuta, E.; Matsukawa, S.; Ebihara, T.; Matsuda, K. Macroion characterization: from dilute solutions to complex fluids; Schmitz, K. S., Ed.; ACS Symposium Series 548; American Chemical Society: Washington, DC, 1994; Chapter 39, p 507. (5) Kokufuta, E. Functional Immobilized Biocatalysts Prepared Using Stimulus-sensitive Polymer Gels. In The Polymeric Materials Encyclopedia - Synthesis, Properties and Applications; Salamone, J. C., Ed.; CRC Press: New York, 1996; Vol. 4, F-G, pp 2615-2621.
the volume of gels through swelling and shrinking. Thus, our first approach involved the control of the gel volume using the difference in the bioaffinities of nonionic and ionic sugar molecules toward lectin (concanavalin A) which had been immobilized in the gel through physical entrapment.6 We also used several enzymatic changes for regulating the volume of the gels: the hydrolysis of ethyl butyrate with esterase,7 the hydrolysis of urea with urease,8 and the oxidation of glucose with glucose oxidase (GOD)9 as well as glucose dehydrogenase (GDH) with the aid of a coenzyme (NAD+).10 In these previous studies, the volume phase transitions in gels were accounted for by hypothesizing a balance between the repulsion and attraction among functional groups attached to the crosslinked polymers which arise from a combination of four intermolecular forces (see ref 11): ionic, hydrophobic, van der Waals, and hydrogen bonding. When a repulsive force, usually electrostatic in nature, overcomes a nonionic attractive force such as hydrogen bonding or hydrophobic interaction, the gel volume should increase discontinuously in some cases and continuously in others. We may expect from previous studies12-15 on polyampholyte gels that the gel would swell at pH levels higher (6) Kokufuta, E.; Zhang, Y.-Q.; Tanaka, T. Nature 1991, 351, 302. (7) Kokufuta, E.; Tanaka, T. Macromolecules 1991, 24, 1605. (8) Kokufuta, E.; Zhang, Y.-Q.; Tanaka, T. J. Biomater. Sci., Polym. Ed. 1994, 6, 35. (9) Matsuda, K.; Orii, H.; Hirata, M.; Kokufuta, E. Polym. Gels Networks 1994, 2, 299. (10) Kokufuta, E.; Aman, Y. Polym. Gels Networks 1997, 5, 439. (11) Ilmain, F.; Tanaka, T.; Kokufuta, E. Nature 1991, 349, 400. (12) Kudaibergenov, S. E.; Sigitov, V. B. Langmuir 1999, 15, 4230. (13) Nisato, G.; Munch, J. P.; Candau, S. J. Langmuir 1999, 15, 4236. (14) Takeoka, Y.; Berker, A. N.; Du, R.; Enoki, T.; Grosberg, A.; Kardar, M.; Oya, T.; Tanaka, K.; Wang, G.-Q.; Yu, X.-H.; Tanaka, T. Phys. Rev. Lett. 1999, 82, 4863. (15) Experimental studies of polyampholyte gels before 1998 are cited in refs 12 and 13; from these references, we may learn a history of polyampholyte gel studies as well as the nature of polyampholyte gels.
10.1021/la001577x CCC: $20.00 © 2001 American Chemical Society Published on Web 04/05/2001
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Table 1. Preparations of Polyampholyte Gels with and without Immobilized Enzymes gel sample
monomer concentration (mg/mL) AAc VI NIPA MBAAm
G1 G2 G2G G2U G2GU
7.0 7.0 7.0 7.0 7.0
9.2 18.4 18.4 18.4 18.4
154.0 154.0 154.0 154.0 154.0
6.6 6.6 6.6 6.6 6.6
enzyme concentration (mg/mL) GOD urease
12.0 0.0 12.0
0.0 2.0 2.0
Figure 1. pH Dependence of normalized equilibrium diameters (d/d0) for polyampholyte gels (G1 and G2) at 37 °C. The normalization of each observed equilibrium diameter (d) was performed with the inner diameter (d0) of the capillary used in the gel preparation.
and lower than an isoelectric point (pI) but shrink at pH ∼ pI. Thus, it is interesting to control the volume of a polyampholyte gel by enzymatically induced pH changes. Such an attempt should help to create a novel biochemomechanical system based on polymer gels. The present paper describes how to design, prepare, and characterize a biochemo-mechanical system consisting of polyampholyte gels that undergo swelling and shrinking changes due to an electrostatic interaction between the opposite charges in the network.
water. The details of the apparatus and measuring procedures have been described in full in our previous papers.10,17
Results and Discussion
Materials. All the monomers were obtained from the commercial sources indicated and used after purification according to the usual ways (e.g., see ref 16): N-isopropylacrylamide (NIPA) from Kojin Chemical Co. (Tokyo, Japan) and acrylic acid (AAc), 1-vinylimidazole (VI), and N,N′-methylenebis(acrylamide) (MBAAm) from Wako Chemical Co. (Tokyo, Japan). Ammonium persulfate (APS, initiator) and N,N,N′,N′-tetramethylethylenediamine (TEMED, accelerator) were also commercial products and used without purification. GOD (EC 1.1.3.4, from Aspergillus niger, with an enzyme activity of 20 000 units/g protein) and urease (EC 3.3.1.5, from Jack beans, with an enzyme activity of 61 000 units/g protein) were purchased from Wako Pure Chemical. Preparation of Gels with and without Immobilized Enzymes. Five gel samples were prepared by mixing the monomers in the proportions shown in Table 1. TEMED (9.36 µL) and APS (1 mg) were added into each of oxygen-free aqueous monomer solutions to initiate the gelation. Then, the reaction was allowed to continue for 1 h at 0 °C using a test tube into which glass capillaries with inner diameters of 0.05 mm had previously been inserted. After the gelation was completed, the gels were taken out of the capillaries and thoroughly washed with distilled water. All the gel samples were cut into cylinders of approximately 2 mm in length and stored at 3 °C before use. Measurements. An aqueous NaCl solution (50 mM, pH 5.42), the pH of which was adjusted with HCl (50 mM), was used as the outer medium in both cases of the presence and the absence of the substrate. Glucose (100 mM) and urea (50 mM) were used as the substrates for GOD and urease, respectively. A gel sample was placed in a micropipet together with either the substrate solution or the substrate-free solution, and its diameter was measured using a microscope (Olympus CK2-TRP-1) with a calibrated scale. The temperature during the measurements was controlled to within 0.01 °C between 20 and 50 °C using circulating
Design of Gels. We focused on a polyampholyte with the fixed charges of the weak-acid and weak-base types whose opposing ionization reactions are dependent on pH. The network obtained via cross-linking of such polymer chains would carry the positive charges at pH < pI because of the protonation of the basic groups, but at pH > pI the network would carry the negative charges due to the deprotonation of the acidic groups. Thus, it is expectable that two pH regions bringing about a swelling change of the network lie on either side of pI, at which a Coulombic attraction among the cross-linked polymer chains becomes greatest to shrink the network. We may employ a combination of AAc with a carboxyl group and VI with an imidazole group for the present purpose. When the network is formed from an equimolar mixture of AAc and VI without use of neutral monomers for lowering the charge density, the gel obtained seems to be in quite a collapsed state over a wide pH region across pI because of a strong intermolecular attraction arising not only from the electrostatic interaction but also from other attractive interactions17 (e.g., hydrogen bonding). Such a collapsed gel does not swell in response to a slight pH change at pH > pI and pH < pI. Our previous study17 has demonstrated that a polyelectrolyte gel of cross-linked poly(ethyleneimine) undergoes a volume collapse at pH 10.7 when pH is increasing, but the collapsed gel does not swell when pH decreases up to pH 5.9. This was due to the formation of stable hydrogen bonding among the cross-linked polymer chains. Taking these into account, we designed the preparation of a polyampholyte gel via terpolymerization of AAc and VI with a neutral monomer (NIPA). By use of the neutral monomer, we intended to reduce the density of the ionic monomer residues in the network. Table 1 shows the mixing ratios of each monomer used in our gel preparation. Effects of pH and Temperature on Gel Swelling. Figure 1 shows the pH dependence of the swelling degree (d/d0) for two polyampholyte gels (G1 and G2). The swelling measurements as a function of pH were made at a fixed temperature (37 °C). We considered that this temperature
(16) Ito, S.; Ogawa, K.; Suzuki, H.; Wang, B.; Yoshida, R.; Kokufuta, E. Langmuir 1999, 15, 4289.
(17) Kokufuta, E.; Suzuki, H.; Yoshida, R.; Yamada, K.; Hirata, M.; Kaneko, F. Langmuir 1998, 14, 795.
Experimental Section
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Figure 2. Temperature dependence of normalized equilibrium diameters (d/d0) for polyampholyte gels (G1 and G2) at pH 5.42.
is close to the optimum temperature for GOD-catalyzed oxidation of glucose as well as for urease-catalyzed hydrolysis of urea. The molar ratios of each monomer in the preparation are NIPA/AAc/VI ) 14:1:1 for G1 and 14:1:2 for G2. As can be seen from Figure 1, there is a characteristic pH at which the swelling curves exhibit a minimum value. Two pH regions (i.e., pH < 5 and pH > 6) leading to a swelling of the gel lie on either side of this characteristic pH. An increase in pH at pH < 5 brings about the shrinking of the gel, whereas the gel swells with increasing pH at pH > 6. At pH > 6, there is little difference in the d/d0 values at the same pH between G1 and G2 because the gels were prepared at the same AAc concentration. Over the whole range at pH < 5, however, the d/d0 value for G2 is about 1.16 times that for G1. This may be attributed to a difference in the VI contents between G1 and G2; that is, the concentration of VI used in the preparation of G2 is two times that for G1 (see Table 1). The results mentioned above clearly indicate that both G1 and G2 behave as a polyampholyte gel with pI near pH 5.5. Gels consisting of cross-linked NIPA homopolymer are well-known to undergo a volume phase transition in response to an infinitesimal change in temperature around 33 °C (e.g., see ref 18). Our ampholyte gels contain NIPA as the neutral monomer; thus, we examined how temperature affects the swelling degree of G1 and G2 (Figure 2). The measurements were made at pH 5.42, the value of which is close to pI. At this pH and at 37 °C, G1 is in a collapsed state (d/d0 ) 0.836) but G2 is in a slightly swollen state (d/d0 ) 0.897) (see Figure 1). Upon being heated above 37 °C, both G1 and G2 shrink still more and reach a fully collapsed state at 54 °C for G1 and at 65 °C for G2. In contrast, on being cooled below 37 °C the gels swell even more. These swelling-shrinking changes were reversible. Therefore, it becomes clear that our ampholyte gels behave as a thermally responsive gel. It is interesting to compare the swelling behavior of our polyampholyte gels with that of the usual NIPA-based polyelectrolyte gels. In general, polyelectrolyte gels consisting of copolymers of NIPA with ionic monomers such as AAc undergo a discontinuous or abrupt volume transition at a certain temperature, referred to as a volume phase transition temperature (TV). An increase in the charge density of the network brings about a rise in TV, accompanying an increase in the volume at TV. In contrast to these general characteristics, our ampholyte gels (18) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163 and references therein.
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exhibited a continuous volume collapse with increasing temperature; this trend is more clear in G2 than in G1. The overall charge due to the COO- and dNH+- ions in G1 seems to be zero at the pH of 5.42 used in the study of temperature effects, because the pI for G1 was in the pH range of 4.8-5.5 (see Figure 1). For G2, however, this pH was lower by 0.5 units than its pI (pH ∼ 5.9); therefore, G2 should have a slight amount of cationic charges. Taking this into account, one may assume the effect of cationic charges to understand why the swelling degree at pH 5.42 for G2 was larger than that for G1 over a wide temperature range, in particular at temperatures >34 °C very near to the TV for neutral NIPA gels. Nevertheless, the continuous change in the swelling degree with temperature, observed not only in G2 but also in G1, may not be explained simply in terms of the charge effect. We have studied in our previous paper19 how inhomogeneous distribution of the COO- ions bound to the NIPA network affects the thermally induced gel collapse. It was suggested that the NIPA chain segments around the locally concentrated charges in the network fail to form hydrophobic aggregates via the collapse transition at temperatures >TV. Then, it is natural to consider the formation of microphases that undergo a collapse transition at different temperatures, so that the gel exhibits a continuous volume change over a wide temperature range. Taking these into account, the continuous swelling curves observed in Figure 2 are understood by assuming the existence of microdomains with locally concentrated charges. This assumption may be supported by the following facts: (i) The plots of d/d0 versus temperature for G1 and G2 bend down around the temperature (34 °C) close to the TV of the pure NIPA gel. (ii) The d/d0 values under a fully collapsed state are 0.55 for G1 (>54 °C) and 0.57 for G2 (>65 °C); these values are larger than that (0.45) for the NIPA-based AAc gels in which COONa groups of 0-18 mol % are randomly distributed (see ref 20). We prepared the polyampholyte gels in the presence of TEMED, so that the copolymerization accompanying the cross-linking reaction should progress at an alkaline pH (∼8). Under such a condition, almost all the AAc monomers are ionized via the dissociation of COOH, whereas the VI monomers are fully deprotonated and neutral. Thus, one might assume that the gelation would occur in a similar way to random copolymerization of NIPA with ionic monomers such as AAc. In the present system in which the anionic and cationic monomers coexist, however, there is the possibility of the formation of AAc-rich and/or VIrich NIPA oligomer chains during the polymerization reaction. This is mainly due to the fact that we cannot perform the gelation with “stirring”, and therefore inhomogeneous polymerization would take place during the cross-linking reaction. As a result, ionic monomer-rich domains are formed within the NIPA network. Such domains should cause the NIPA segments to fail in the formation of hydrophobic aggregates, even at a pI at which the overall charge is zero but parts of the AAc and VI monomers in the network are ionized. To support this (19) Kokufuta, E.; Wang, B.; Yoshida, R.; Khokhlov, A. R.; Hirata, M. Macromolecules 1998, 31, 6878. (20) It has been found that the d/d0 values at a fully collapsed state for NIPA gels with randomly copolymerized AAc (as sodium salt) are independent of the charge density and approach a constant value of ∼0.45 upon heating: Hirotsu, S.; Hirokawa, Y.; Tanaka, T. J. Chem. Phys. 1987, 87, 1392. Therefore, the result in (ii) means that there are hydrophilic domains with a number of the COO- and dNH+- ions within G1 or G2. This aspect is very similar to that of NIPA gels into which sodium poly(acrylic acid) was introduced to form a locally concentrated charge domain within the network (see ref 19).
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Figure 3. Kinetics of gel swelling at 37 °C for GOD-loaded G2 (i.e., G2G) in 50 mM NaCl solution containing 100 mM glucose as the substrate. The initial pH of the solution was adjusted to 5.42.
Figure 4. Kinetics of gel swelling at 37 °C for urease-loaded G2 (i.e., G2U) in 50 mM NaCl solution containing 50 mM urea as the substrate. The initial pH of the solution was adjusted to 5.42.
assumption, one may suggest that the swelling measurements under a high ionic strength would be useful. However, the swelling of neutral NIPA gels is strongly affected by the concentration and species of small salts added into the outer solution;21 therefore, we did not perform such experiments for our polyampholyte gels. Time-Dependent Changes in the Size of Polyampholyte Gels Induced by Immobilized Enzyme Reactions. We chose G2 for the construction of biochemomechanical systems because G2 clearly exhibited pI in a very narrow pH range compared with G1 (see Figure 1). At first G2G and G2U were prepared by a single immobilization of GOD and urease into G2, respectively (see Table 1). Urease catalyzes the hydrolysis of urea into ammonia and carbon dioxide;
of the substrate just as we had expected. The swelling degree appeared to level off within 1 h for G2U and within 7 h for G2G. The d/d0 value after it leveled off (i.e., d/d0 ) 1.07 for G2G and 1.03 for G2U) was compared with the d/d0 versus pH curve for G2 in Figure 1, to estimate the pH level within the gel under a fully swollen state which had enzymatically been attained. This comparison gave us the following data: pH ∼ 3.2 for G2G and pH > 9 for G2U. Our preliminary experiments with the “free” enzymes revealed that the solution pH after the reactions in eqs 1 and 2 reached a steady state was 3.2 in the GOD system and 9.2 in the urease system. These pH values agreed with the estimated values of pH within the gel phase. Thus, it may be said that the enzymatic pH change within the gel phase occurs without a serious loss in the immobilized enzyme activity. In the case of G2U, a slight shrinkage was observed immediately after the addition of urea (see Figure 4). This is due to the pH (5.42) of the substrate solution at the beginning of the immobilized enzyme reaction. An increase in pH within the gel should allow G2U to shrink until the pH level reaches pI. Thus, a minimum d/d0 value (∼0.865) observed in Figure 4 is comparable to the swelling degree (∼0.862) for G2 at pI (see Figure 1). One might be interested in discussing why G2G exhibited a slow swelling kinetics compared with G2U. We have dealt with this question in our previous papers10 and found that the main reason was the formation of D-gluconic acid. As shown in eq 2, the gluconic acid is formed thermally, but not enzymatically, from D-gluconoδ-lactone (neutral) as the product of the enzyme reaction. In addition, an increase in the gluconic acid concentration within the gel phase should promote the formation of D-glucono-γ-lactone. As a result, the generation rate of H+ ions in the GOD system is slower than that of OHions in the urease system. Enzymatically Induced Shrinking-Swelling Oscillation in Polyampholyte Gel with Coimmobilized GOD and Urease. The most interesting property of the present polyampholyte gels would be the pH change in their swelling degree at a given temperature; that is, a swollen gel shrinks and swells again when increasing or decreasing pH passing through pI. Enzymatic raising and lowering of pH within the gel phase should be possible when GOD and urease are coimmobilized. Figure 5 demonstrates typical data for swelling-shrinking oscillation in G2GU. Initially, the gel swells upon the addition
(NH2)2CdO + 3H2O f CO2 + 2NH4OH
(1)
both products immediately react with water molecules to raise the pH of the reaction system.22 In contrast, GOD decreases the solution pH because of the oxidation of glucose to form gluconic acid as follows:23 GOD
∆
β-D-glucose 98 D-glucono-δ-lactone y\z D-gluconic
∆
acid y\z D-glucono-γ-lactone (2)
Thus, it is expected that when glucose is added into the outer medium at pH near pI, G2G would swell because of the protonation of imidazole groups. Also expected is the swelling of G2U upon the addition of urea because of the dissociation of the COOH groups. Figures 3 and 4 show the swelling kinetics of G2G and G2U, respectively. Both gels were incubated at 37 °C in 50 mM NaCl solution at pH 5.42, the solvent of which was the same as that used in the study of the temperature dependence. After the incubation was allowed to continue for 1 h, a very slight volume of the substrate solution containing a high concentration of glucose or urea was quickly injected into the outer medium by means of Ar gas pressure. The substrate concentration at the beginning for the measurements was adjusted to 100 mM for glucose and 50 mM for urea. The gel swelled upon the addition (21) Suzuki, A. Adv. Polym. Sci. 1993, 110, 199. (22) Reithel, F. J. Ureases. In The Enzymes, 3rd ed.; Boyer, P. D., Ed.; Academic Press: New York, London, 1971; Vol. 4, pp 1-21. (23) Bentley, R.; Neuberger, A. Biochem. J. 1949, 45, 584.
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Figure 5. Shrinking-swelling oscillation in G2GU (i.e., G2 with coimmobilized GOD plus urease) upon alternative addition of urea and glucose. The swelling at first run was started in the same way as used for G2G. G and U denote the addition of glucose (100 mM) and urea (50 mM), respectively.
of glucose into the outer medium at pH 5.42. After the attainment of a fully swollen state (d/d0 ∼ 1.06), urea was added into the outer medium. This leads the gel to shrink until the level at pI (d/d0 ∼ 0.86) and then to swell again. Because the observed shrinking-swelling cycle upon the addition of urea (second run) should be due to the increase in pH induced by the immobilized urease, further addition of glucose brings about a new shrinking-swelling cycle (third run) due to the GOD-induced decrease in pH. When both enzyme reactions are repeated in a closed system, salts such as ammonium gluconate and ammonium
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bicarbonate should be accumulated. This would strengthen the buffering capacity of the solution; therefore, the rate of decrease in pH as well as increase in pH becomes slow by repetition of both enzyme reactions. Therefore, the time taken for the gel to swell became slower in G2GU than in G2G or G2U (see second run with urea and third run with glucose). It is difficult to theoretically describe the aspect for enzymatically induced shrinking-swelling oscillation in G2GU. For example, let us consider pH change only within the gel phase. The acid is formed enzymatically, but at the same time it is diffused into the bulk phase. In addition, the acid formed within the gel phase should be neutralized with bases coming from the bulk phase. These cause a pH gradient within the gel phase. Therefore, the rate of the enzymatically induced decrease or increase in pH becomes a function of time and position within the gel. The actual system seems to be more complicated, and at present there is no good theoretical approach for analyzing such a system. However, the idea that the coimmobilization of GOD plus urease in polyampholyte gels enables the shrinking-swelling oscillation by alternative uses of the two substrates, as demonstrated here, would provide a guiding principle in technological applications of polyampholyte gels in the future. Acknowledgment. This work was supported in part by a Grant-in-Aid for Scientific Research to E.K. from the Ministry of Education, Japan (No. 08558092). LA001577X
