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pH Control of Transport through a Porous Membrane Self-Assembled with a Poly(acrylic acid) Loop Brush Hongjie Zhang and Yoshihiro Ito* Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Tokushima 770-8506, Japan Received April 26, 2001. In Final Form: August 7, 2001 Side chains of poly(acrylic acid) (PAA) were conjugated with cysteamines for self-assembly on a goldcoated membrane. The self-assembly of the cysteamine-modified PAA on the membrane was monitored by surface plasmon resonance spectroscopy. It was found that the amount of assembled polyelectrolytes significantly depended on the concentration of the polyelectrolytes, the content of cysteamine in the polyelectrolyte, solution pH, and ionic strength. Transport through porous membranes gold-coated and subsequently self-assembled with the polyelectrolyte was investigated. Water permeability through the membrane was reversibly regulated by pH and ionic strength. The permeability was high at low pH and low at high pH, and an increase in ionic strength increased the permeability at high pH. It was suggested that at low pH the polyelectrolyte was protonated which caused the brushes to shrink and opened the pores. At high pH, the opposite process took place. The ionic strength reduced the electrostatic repulsion between the ionized polyelectrolytes that shrank the opening of the pores. These phenomena depended on the state of the self-assembled polyelectrolytes. Filtration of water solutions of ionic (oligodeoxyribonucleotide) and nonionic (poly(ethylene glycol)) polymers through the modified membrane was also studied. pH-responsive permeability depended on the molecular weight of the solutes.
Introduction Polymer brushes attracted polymer chemists’ attention in the 1950s, when it was found that grafting polymer molecules to colloidal particles was a very effective way to prevent flocculation.1 It was found that polymer brushes can be useful in other applications such as new adhesives, protein-resistant biosurfaces, chromatographic media, lubricants, polymer surfactants, and polymer compatibilizers.1 Using polymer brushes, some researchers have designed nanoporous membranes or synthetic transmembrane channels to mimic biological functions.2 To prepare an environmentally responsive membrane, polyelectrolyte brushes have been used.1,2 Transport through these membranes can be regulated by pH, ionic concentration, temperature, photoirradiation, oxidoreduction reaction, and glucose concentration.1-14 According to Zhao and Brittain,1 the preparation of polymer brushes can be classified into two types depending on whether they are “grafting to” or “grafted from”. The former refers to preformed, end-functionalized polymers (1) Zhao, B.; Brittain, W. J. Prog. Polym. Sci. 2000, 25, 677-710. (2) Ito, Y.; Park, Y. S. Polym. Adv. Technol. 2000, 11, 136-144. (3) Yamaguchi, T.; Ito, T.; Sato, T.; Shindo, T.; Nakao, S. J. Am. Chem. Soc. 1999, 121, 4078-4079. (4) Hou, Z.; Abbot, N. L.; Stroeve, P. Langmuir 2000, 16, 2401-2404. (5) Ito, Y.; Park, Y. S.; Imanishi, Y. Langmuir 2000, 16, 5376-5381. (6) Peng, T. Ph.D. Thesis, University of Toronto, 1999. (7) Hulteen, J. C.; Jirage, K. B.; Martin, C. R. J. Am. Chem. Soc. 1998, 120, 6603-6604. (8) Castro, P. C.; Cohen, Y.; Monbouquette, H. G. J. Membr. Sci. 1993, 84, 151-160. (9) Liu, Y.; Zhao, M.; Bergbreiter, D. E.; Crooks, R. M. J. Am. Chem. Soc. 1997, 119, 8720-8721. (10) Jimbo, T.; Ramirez, P.; Tanioka, A.; Mafe, S.; Minoura, N. J. Colloid Interface Sci. 2000, 225, 447-454. (11) Minagawa, M.; Tanioka, A.; Ramirez, P.; Mafe, S. J. Colloid Interface Sci. 1997, 188, 176-182. (12) Minagawa, M.; Tanioka, A. J. Colloid Interface Sci. 1998, 202, 149-154. (13) Chen, J.; Minoura, N.; Tanioka, A. Polymer 1994, 35, 28532856. (14) Nishizawa, M.; Menton, V. P.; Martin, C. R. Science 1995, 268, 700-702.
reacting with suitable surface functionalities to form a tethered polymer brush. The latter refers to initiators that are immobilized onto the surface followed by in situ surface-initiated polymerization to generate tethered polymers. The former approach is useful to regulate the polymer length and surface concentration and is used by some researchers.15-22 Recently, a self-assembly technique, which is one of the simplest and most effective methods to prepare thin films, was employed for fabricating a stimulus-responsive filter.4 Self-assembled monolayers of thiol-functionalized carboxylic acid were deposited on gold to turn a porous membrane into a pH-dependent valve. Ito et al. demonstrated that a polyelectrolyte chain (poly(L-glutamic acid)) end-grafted to a nanoporous membrane and the membrane pores could open and close in response to pH, working as a gate.5 In the present study, poly(acrylic aid) with thiolmodified chains was prepared and the modified polyelectrolyte was self-assembled on a gold-coated surface. The self-assembly behavior was monitored by surface plasmon resonance (SPR), and molecular transport through the modified porous membrane was investigated. Materials and Methods Synthesis of Modified PAA. Poly(acrylic acid) (PAA, Mw ) 30 000, Aldrich) was derivatized by thiol in the side chains (15) Koutsos, V.; Van der Vegte, E. W.; Hadziioannou, G. Macromolecules 1999, 32, 1233-1236. (16) Koutsos, V.; Van der Vegte, E. W.; Pelletier, E.; Stamouli, A.; Hadziioannou, G. Macromolecules 1997, 30, 4719-4726. (17) Bergbreiter, D. E.; Franchina, J. G.; Kabza, K. Macromolecules 1999, 32, 4993-4998. (18) Yang, X.; Shi, J.; Johnson, S.; Swason, B. Langmuir 1998, 14, 1505-1507. (19) Ebata, K.; Furukawa, K.; Matsumoto, N. J. Am. Chem. Soc. 1998, 120, 7367-7368. (20) Wells, M.; Crooks, R. M. J. Am. Chem. Soc. 1996, 118, 39883989. (21) Tran, Y.; Auroy, P.; Lee, L.-T. Macromolecules 1999, 32, 89528964. (22) Prucker, O.; Naumann, C. A.; Ruhe, J.; Knoll, W.; Frank, C. W. J. Am. Chem. Soc. 1999, 121, 8766-8770.
10.1021/la0106079 CCC: $20.00 © 2001 American Chemical Society Published on Web 11/30/2001
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Figure 1. Schematic representation of the synthesis of poly(acrylic acid)-carrying thiol groups at the side chains (a) and selfassembly on a gold-coated porous membrane (b). according to scheme in Figure 1a. The oxidation of cysteamine (Wako Pure Chemical Industry, Ltd., Osaka, Japan) was performed by incubation at pH 8.5 for 48 h. The thiol content of the product was estimated from the absorbance at 412 nm of 3-carboxylato-4-nitro thiophenoylate ions produced from the Ellman reaction of 5,5′-dithiobis(2-nitro-benzoic acid) (Nacalai Tesque, Kyoto, Japan) with the thiol groups.23 After 48 h, the conversion reached 99.5%. The product was added to a solution containing 0.18 g of PAA in 20 mL of Milli-Q water, and the pH of the mixture was adjusted to 6 with HCl. The mixture was kept at room temperature and continuously stirred. Subsequently, 240 mg of 1-ethyl-3-(3dimethyl amino-propyl) carbodiimide (water-soluble carbodiimide, WSC) was added in portions of 24 mg at 10 min intervals. The reaction mixture was kept at pH 6 by adding HCl. After addition of WSC, the stirring was continued at 4 °C for 48 h. The resulting polymer was hydrolyzed by adjusting the pH to 10, and the disulfide dimers grafted onto the PAA were then reduced to a thiol group by 12 h treatment with (()dithiothreitol (DTT, Wako Pure Chemical Industry, Ltd., Osaka, Japan). To remove excessive reactants, ultrafiltration was conducted using a seamless cellulose tube (Amicon model, Millipore UFP1 LCC 24, with a cutoff molecular weight of 5000). The purified polymer was lyophilized and referred to as PAA-SH. The content of cysteamine in PAA-SH was determined by quantitative measurement of thiol groups. The content of thiol was measured using the Ellman reaction as described above. Self-Assembly of PAA-SH. The synthesized PAA-SH was self-assembled on the surface of a gold-coated nanoporous membrane, as shown in Figure 1b. The preparation of the goldcoated porous membrane and the self-assembly method were the same as previously reported.5 A porous polycarbonate (PC) membrane (Dupont nucleopore membrane; average pore diameter, 200 nm) was coated with gold in an E-1010 ion sputter (Hitachi Co., Hitachi, Japan). The thickness of the gold was adjusted to ca. 50 nm. The coated membrane was exposed to an aqueous solution of PAA-SH (0-60 × 10-3 mol/L) at different pHs and ionic concentrations for 24 h. The surface-modified membrane was rinsed with Milli-Q water until the pH of the washing liquid became neutral. SPR was measured using an instrument (BIAcore Co., Ltd., Sweden) to determine the amount of PAA-SH assembled on the cover glass (22 × 22 mm; Matsunami Glass Ind., Ltd., Osaka, Japan) onto which gold film (approximately 50 nm thick) was vapor-deposited in the same way as that used for the PC membrane. Transport through the Membrane. Transport through the membranes assembled with the PAA-SH was investigated using (23) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70-75.
a centrifugal filter device (Microcon, YM-50, Millipore, U.S.A.). The prepared membrane was mounted on a filter reservoir containing a cellulose membrane attached to a vial for filtrate collection. The reservoir was filled with a certain amount of aqueous solution. The pH of the solution was adjusted to the demanded value using NaOH and HCl. The ionic strength was adjusted by NaCl as estimated by ([Na+] + [Cl-])/2. The aqueous solution was allowed to flow under a constant centrifugal force (1710g). The flux J through the membrane was calculated according to the following equation.
J ) V/St where V is the volume (mL) of transported fluid, S is the active surface area (cm2) of the membrane, and t is the time (h) span of transport collection. The volume of fluid transported through the membranes linearly increased with time. To determine macromolecular transport, poly(ethylene glycol) (PEG) (4.4 mg/mL) and DNA (4 × 10-4 mg/mL) solutions were prepared. After the transport experiments, the concentration of transported PEG was monitored by SPR. The calibration was performed by measuring the SPR responses of known concentrations of PEG. On the other hand, DNA transport was monitored by measuring its UV absorbance at a wavelength of 260 nm. The calibration was carried out by measuring the absorbance of known concentrations of DNA.
Results and Discussion Membrane Preparation. The thiol content in PAASH increased with increasing reactant concentration which allowed for the simple regulation of the thiol group content in the polyelectrolyte (see Supporting Information). To confirm the self-assembly of PAA-SH on the gold surface, SPR measurements were performed using PAASH and PAA. After addition of polyelectrolyte solution, the gold surface was washed with pure water. Although slight physical adsorption of PAA was observed, significant binding of the PAA-SH to the gold surface occurred (Figure 2), which confirmed the self-assembly of PAASH through the thiol groups present in the polymer. The amount of assembled PAA-SH was calculated from the change in the resonance signal before and after the gold surface was exposed to the PAA-SH solution. It was reported that in the case of protein adsorption 1000 resonance units of SPR response corresponds to a surface concentration of about 1 ng/mm2 independently of mo-
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Figure 5. Water permeability through membranes selfassembled with PAA-SH. The surface densities of selfassembled PAA-SH were zero (O), 4.5 pmol/cm2 (9), 18 pmol/ cm2 (b), and 25 pmol/cm2 (2). Figure 2. SPR response of PAA and PAA-SH on a gold-coated plate.
Figure 6. Water permeability through membranes selfassembled with PAA-SH at ionic strength 0 (b) and 1 (9). The surface density of self-assembled PAA-SH was 18 pmol/cm2. Figure 3. Self-assembly of PAA-SH having different contents of thiol groups onto a gold-coated plate at pH 3 and zero ionic strength. The thiol content was 2 mol % (9), 10 mol % (b), and 30 mol % (2). The mol % means thiol molecules/(carboxylic acid units in PAA-SH) × 100.
Figure 7. Water permeability through membranes selfassembled with PAA-SH containing different amounts of thiol. The surface density of self-assembled PAA-SH was 12 pmol/ cm2.
be in the order of 10 pmol/cm2. It was reported that the amount of self-assembled polypeptide, DNA, and alkane Figure 4. Self-assembly of PAA-SH (thiol content, 2 mol %) at pH 3 and ionic strength 0 (9), pH 7 and ionic strength 1 (b), and pH 7 and ionic strength 0 (2).
lecular size.24 This calibration was used to characterize the adsorption of various polymers.25 Taking this into consideration, the amount of PAA-SH was estimated to (24) Stenberg, E.; Persson, B.; Roos, H.; Urbaniczky, C. J. Colloid Interface Sci. 1991, 143, 513-526.
(25) Lo¨fås, S.; Johnsson, B.; Tegendal, K.; Ro¨nnberg, I. Colloids Surf., B 1993, 1, 83-89. (26) Herbert, C. B.; Mclernon, T. L.; Hypolite, C. L.; Adams, D. N.; Pikus, L.; Huang, C. C.; Fields, G. B.; Letourneau, P. C.; Distefano, M. D.; Hu, W. S. Chem. Biol. 1997, 4, 731-737. (27) Jodan, C. E.; Frutos, A. G.; Thiel, A. J.; Corn, R. M. Anal. Chem. 1997, 69, 4939-4947. (28) Peterlinz, K. A.; Georgiadis, R. M.; Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 3401-3402. (29) Frey, B. L.; Jodan, C. E.; Kornguth, S.; Corn, R. M. Anal. Chem. 1995, 67, 4452-4457.
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Figure 8. Illustration of pH-dependent permeation through a membrane self-assembled with PAA-SH containing different amounts of thiol. Table 1. PEG Transporta b
Mw
λ1 (nm)
600 8000 20000 500000
4 55 138 3438
R
(%)c
at pH ) 2 0 0 100 100
R
(%)c
at pH ) 7 0 33 100 100
a The amount of self-assembled PAA-SH is 18 pmol/cm2. b λ is 1 the end-to-end length of the PEG macromolecule and is calculated by considering the macromolecule in an extended state (ref 31).c R is polymer retention, which is defined as R ) (1 - Cp/C0) × 100%, where C0 is the concentration of polymer in filtered solution and Cp is the concentration of polymer in permeate.
Table 2. DNA Transporta DNA length (base)
λ2b (nm)
20 103 1000
6-8 30-40 300-400
R (%) at pH ) 2 R (%) at pH ) 7 0 0 100
0 37 100
a The amount of self-assembled PAA-SH is 18 pmol/cm2. b λ is 2 persistent length (ref 32).
was in the range of 1-100 pmol/cm2.5,26-29 The amount of self-assembled PAA-SH was in that range. The amount of self-assembled PAA-SH depended on the concentration of PAA-SH in solution and the content of thiol in PAA-SH. Figure 3 shows that the surface density of PAA-SH increased with an increase in concentration and was saturated at a certain concentration. Figure 3 also shows that the saturated amount decreased with the increase of thiol content of PAA-SH. By calculation of the surface density of PAA-SH, the saturated densities were 25, 14, and 4.5 pmol/cm2 for 2, 10, and 30 mol % (thiol molecules/(acrylic acid unit in PAA-SH) × 100), respectively. The increase of thiol content led to an increase in the binding sites and thus increased the occupation of binding sites on the surface. The loop structure of PAA-SH increased the area of PAASH-gold binding by the steric hindrance. The amount of self-assembled PAA-SH significantly depended on the pH of solution (Figure 4). High pH decreased the amount of self-assembled PAA-SH. Potentially, at high pH PAA-SH had an extended structure and increased its coverage on the surface, which decreased the saturated surface density. Finally, the increase of ionic strength of the adsorption solution increased the saturation surface density. This was due to the reduction of electrostatic repulsion of side chains of PAA-SH, which reduced the extended structure.
Water Transport. The rate of water transport through the bare membrane was independent of pH, whereas the transport through the grafted membrane depended upon pH (Figure 5). High permeability was observed at low pH, while low permeability was seen at neutral pH. It was considered that in the region of low pH the PAA-SH chain was protonated and existed in a tight globular state to open the pores; on the other hand, in the region of high pH it was deprotonated to form an extended structure to cover the pores. Kontturi et al.30 proposed a simple theoretical model describing the effects of pH on permeability and validated it experimentally for the case of poly(vinylidene fluoride) membranes graft-modified with a small amount of PAA. The transport experiments were repeated from low to high pH and from high to low pH several times, and the results were reproducible. The binding between PAASH and the gold was stable under the experimental conditions. The membrane response was related to the amount of assembled PAA-SH (Figure 5). Low surface densities of PAA-SH did not significantly affect the permeability, while a high surface density of PAA-SH reduced the water permeability and its pH dependence. A moderate surface density of assembled PAA-SH showed the best sensitivity to the environmental conditions. Ionic strength also affected the transport rate of water (Figure 6). At high pH conditions, permeability was strongly dependent on ionic strength. However, the effect was quite small under low-pH conditions. At a high ionic strength, the water transport became less sensitive to changes in pH. Our guess is that a high ionic concentration shielded deprotonated polymers from electrostatic repulsion. The water transport through the membranes selfassembled with PAA-SH significantly depended on the state of graft chains at a constant surface density of 12 pmol/cm2 (Figure 7). The pH sensitivity was low for the membrane self-assembled with PAA-SH that had a higher content of thiol groups. It was considered that the increase of thiol content increased the binding sites on the gold surface, thus leading to a compressed state of self-assembled polymers. The molecular flexibility of selfassembled PAA-SH with a high thiol content should be (30) Kontturi, K.; Mafe, S.; Manzanares, J. A.; Svarfvar, B. L.; Viinikka, P. Macromolecules 1996, 29, 5740-5746. (31) He, M.; Chen, W.; Dong, X. Polymer Physics; Fudan University Press: Shanghai, 1990. (32) Oana, H.; Yoshikawa, K. Protein, Nucleic Acid Enzyme 1995, 140, 1558-1563.
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lower than that of self-assembled PAA-SH with a low thiol content. Therefore, the pH sensitivity was reduced in the higher content of thiol-gold binding sites. The explanation is illustrated in Figure 8. Polymer Solution Transport. DNA and PEG with different molecular weights were used for a transport through PAA-SH-modified membranes. Polymer retention (R) shown in Tables 1 and 2 indicates that there was almost no solute transport either at pH 2 or at pH 7 when the molecular weight exceeded 20 000 for PEG and 1 kb for DNA. On the other hand, for low molecular weight PEG and DNA all molecules could be transported through the membrane. However, the R value of PEG 8000 and DNA 103 base was low at pH 2 and high at pH 7. The transport of macromolecules with a moderate molecular weight depended on pH. It was considered that the macromolecule size was so close to the pore size that the transport was regulated by pH-dependent conformational change of the polymer brush on the pore.
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Conclusions The present study demonstrated the characteristic sideon adsorption of a polyelectrolyte onto a porous membrane and the associated water and polymer permeability dependence on the adsorbed state of the polyelectrolyte. Using this self-assembly process, the solute transport was regulated by the structure of the adsorbed polyelectrolyte. Acknowledgment. Y. Ito thanks the Mukai Science and Technology Foundation and the Ministry of Education, Culture, Sports, Science, and Technology of Japan (11167254) for financial support. The authors thank Dr. K. E. Healy at the University of California, Berkeley, for his valuable comments throughout the manuscript. Supporting Information Available: Relationship between feed of cysteamine and thiol content in PAA-SH. This material is available free of charge via the Internet at http://pubs.acs.org. LA0106079
