ARTICLE pubs.acs.org/IECR
Temperature-Swing Adsorption of Proteins in Water Using Cationic Copolymer-Grafted Silica Particles Shintaro Morisada,*,† Ken-ichiro Namazuda,† Shitoka Suzuki,† Noriko Kikuchi,† Haruka Kanda,† Yoshitsugu Hirokawa,‡ and Yoshio Nakano† †
Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8502, Japan ‡ Department of Materials Science, The University of Shiga Prefecture, 2500 Hassaka, Hikone, Shiga 522-8533 Japan ABSTRACT: We have prepared silica particles grafted with poly(N-isopropylacrylamide) (PNIPA) copolymers as adsorbent for the temperature-swing adsorption of bovine serum albumin (BSA) in water, where vinylbenzyl trimethylammonium chloride (VBTA), 2-(dimethylamino)ethyl methacrylate (DMAEMA), or N,N-dimethylacrylamide (DMAA) was employed as a comonomer. The surface potentials of PNIPA-grafted and P(NIPA-co-DMAA)-grafted silica particles in water at 298 and 313 K were negative, while those of P(NIPA-co-VBTA)-grafted silica particle were positive, because VBTA is a quaternary ammonium salt and positively charged in aqueous solutions. As for the P(NIPA-co-DMAEMA)-grafted silica particle, the surface potential changed from positive to near zero with increasing temperature from 298 K to 313 K. This may be because the coil-to-globule transition of grafted copolymers leads to the dehydration and deprotonation of DMAEMA group, which is a tertiary amine and can be positively charged only in the aqueous phase. Although PNIPA-grafted and P(NIPA-co-DMAA)-grafted silica particles failed to adsorb BSA, P(NIPAco-VBTA)-grafted and P(NIPA-co-DMAEMA)-grafted silica particles adsorbed BSA, indicating that BSA is adsorbed by the electrostatic attraction between the negatively charged BSA and the positively charged copolymers on the silica surface. Moreover, P(NIPA-co-VBTA)-grafted and P(NIPA-co-DMAEMA)-grafted silica particles repeatedly adsorbed BSA at 298 K and desorbed some of the preadsorbed BSA at 313 K via the temperature-swing operation. This BSA desorption may result from the decrease in the number of the positively charged groups accessible to BSA due to the coil-to-globule transition of the grafted copolymers with increasing temperature.
1. INTRODUCTION In recent years, the synthesis and the application of temperature-responsive polymer materials, such as polymer gels and polymer-grafted particles, have been of great interest for researchers and engineers in many fields. Most of these materials are based on poly(N-isopropylacrylamide) (PNIPA), which exhibits a reversible phase transition in water at its lower critical solution temperature (LCST) of ∼306 K.1 Because of the fact that the LCST of PNIPA is approximately room temperature, temperature-responsive materials that utilize PNIPA have been employed, especially in biomedical and biotechnological applications, such as drug delivery,24 cell culture system,57 tissue engineering,8 and chromatographic bioseparation.7,9 It is well-known that adsorption phenomena play an important role in separation and purification processes. In adsorption processes, if a temperature-responsive polymer material is utilized as adsorbent, simplification of the process and reduction of waste can be achieved, because reversible adsorption and desorption might be possible only by temperature change without an addition of any chemicals. Kawaguchi et al.10 studied the adsorption of proteins onto the N-isopropylacrylamide (NIPA) gel at 298 and 313 K—that is, below and above the LCST—and found that the adsorption amount of proteins at 313 K was larger than that at 298 K, because the protein adsorption by the NIPA gel was mainly driven by the hydrophobic interaction between them. In general, the protein adsorption onto a solid surface is determined mainly by the electrostatic and hydrophobic interactions between them and the structural stability of the protein r 2011 American Chemical Society
molecule, and it is well-known that the protein desorption from the solid surface is very difficult to realize.1114 However, they also reported that the protein desorption from the NIPA gel can be realized only by increasing temperature from 298 K to 313 K after the proteins were adsorbed onto the gel at 298 K. To our knowledge, this is the first report on the adsorption and desorption of protein using the NIPA-based material. Although some other research groups have also conducted the protein adsorption experiments using the NIPA-based materials,1523 there have been no reports on protein desorption only by temperature control, except for the above-mentioned study. Previously, we investigated the adsorption and desorption behavior of bovine serum albumin (BSA) in water using the NIPA-based gels copolymerized with vinylbenzyl trimethylammonium chloride (VBTA) or 2-(dimethylamino)ethyl methacrylate (DMAEMA), and found that both cationic copolymer gels can adsorb BSA at 298 K and desorb some of the preadsorbed BSA at 313 K repeatedly via temperature-swing operation.24 The BSA adsorption and desorption mechanisms were considered as follows: (i) the cationic gels adsorbed BSA at 298 K through the electrostatic attraction between the negatively charged BSA and the positively charged VBTA (or DMAEMA) groups in the gel; (ii) the preadsorbed BSA was desorbed by Received: April 12, 2011 Accepted: October 5, 2011 Revised: August 30, 2011 Published: October 05, 2011 12358
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Figure 1. Chemical structures of the monomers: (a) N-isopropylacrylamide (NIPA); (b) vinylbenzyl trimethylammonium chloride (VBTA); (c) 2-(dimethylamino)ethyl methacrylate (DMAEMA); and (d) N,Ndimethylacrylamide (DMAA).
increasing the temperature from 298 K at 313 K, because the number of the positively charged groups accessible to BSA decreased as a result of the shrinking of the copolymer gels above the LCST. Unfortunately, however, the desorption amount of BSA was fairly small, compared to its adsorption amount: in other words, a considerable amount of BSA remained to be adsorbed at higher temperature. For the temperature-swing adsorption, the desorption amount is essential, which practically corresponds to the absolute difference between the adsorption amounts at lower and higher temperatures. In the case of the BSA adsorption onto the gels, the adsorbed BSA exists inside the polymer network of the gel as well as on the gel surface, and the BSA inside the gel would be hardly desorbed. This indicates that the BSA desorption amount could be increased by employing the polymer-based adsorbent without a three-dimensional polymer network. In the present study, therefore, we prepare NIPA copolymergrafted silica particles instead of NIPA copolymer gels as adsorbent for the temperature-swing adsorption of BSA in water. In addition to two cationic monomers, VBTA and DMAEMA, we also employ a hydrophilic and neutral monomer, N,Ndimethylacrylamide (DMAA), as a comonomer in the grafted polymer for comparison. We estimate the size of the grafted polymer on the silica surface and investigate the temperature dependence of the surface potential of the polymer-grafted silica particles. Subsequently, we perform the temperature-swing adsorption experiments and discuss the adsorption and desorption behaviors of BSA on and from the polymer-grafted silica particles prepared here.
2. EXPERIMENTAL SECTION 2.1. Materials. NIPA and DMAA were kindly provided by Kojin Co., Ltd. (Tokyo, Japan), and VBTA and DMAEMA were purchased from Aldrich (St. Louis, MO) and Wako Pure Chemical Industries, Ltd. (Osaka, Japan), respectively. The chemical structures of these monomers are displayed in Figure 1. 2,20 -Azobisisobutyronitrile (AIBN) and 3-methacryloxypropyl trimethoxysilane (MPTS) were obtained from Wako Pure Chemical and Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan), respectively. Hollow silica particles (8 μm in average diameter) were kindly provided by Fuji Silycia Chemical, Ltd. (Aichi, Japan). To obtain the BrunauerEmmettTeller (BET) surface area of the silica particles (SBET), we measured nitrogen adsorptiondesorption isotherms at 77 K with an automated gas sorption analyzer (OMNIXORP 100CX, Beckman Coulter, Brea, CA) and found SBET to be equal to 3.1 m2/g. BSA was purchased from Aldrich. Deionized and distilled water was used in all procedures, and all reagents were used as received.
Figure 2. Synthesis of polymer-grafted silica particles using 3-methacryloxypropyl trimethoxysilane (MPTS).
2.2. Preparation of Polymer-Grafted Silica Particles. In the present study, four types of polymer-grafted silica particles were prepared, as shown in Figure 2: MPTS, which has the vinyl group, was immobilized on the silica surface according to the procedure reported by Philipse and Vrij,25 and then the free radical polymerization was conducted using the MPTS-immobilized silica particles (MPTS-Si) and the monomer(s). Note that although MPTS forms a multilayer a few nanometers thick on the silica surface,25 we simplified the illustration in Figure 2. Silica particles (25 g) were washed with 1.0 L of 1.0 wt % HCl aqueous solution at room temperature for 12 h and then rinsed with water, followed by drying at 383 K for 12 h. The dried silica particles were added to a mixture solution of ethanol (440 mL) and aqueous ammonia (25 wt %, 27 mL), and the solution was stirred at room temperature for 2.5 h. MPTS (20 mL) was added to the solution, and after 1 h, ∼250 mL of solvent was distilled away under reduced pressure. The MPTS-Si was washed thoroughly with ethanol and then dried at 323 K for 12 h. The graft polymerization using MPTS-Si was conducted as follows. The monomer(s) and AIBN (initiator, 0.046 g) were dissolved in 200 mL of ethanol: the total monomer concentration was 1.0 M, and the molar ratio of NIPA to comonomer was 95:5 in the preparation of copolymer-grafted silica particles. After addition of MPTS-Si (10 g), the solution was deoxygenated by nitrogen bubbling for 1 h, and then the polymerization was carried out under nitrogen atmosphere with stirring at 333 K for 6 h. The polymer-grafted silica particles were separated from the solution by filtration under reduced pressure. The polymergrafted silica particles were washed with a large volume of ethanol to remove residual chemicals and rinsed with water. For further washing, the polymer-grafted silica particles were added to a vial containing 45 mL of water. The vial was well-sealed and shaken at room temperature for 12 h, and then the particles were collected by filtration. This washing procedure was repeated until the 12359
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Table 1. Properties of Polymer-Grafted Silica Particles surface potential, ψe [mV]
sample name
NIPA/comonomera (molar ratio)
LCSTb [K]
amount of grafted polymers, grafted polymer density, grafted polymer wc [mg-polymer/g-Si] Fpd [chains/nm2] distance, d [nm]
306
11.0
at 313 K
70 ( 8.2
75 ( 9.6
0.0497
4.49
35 ( 9.4
73 ( 8.2
silica particle PNIPA-Si
at 298 K
85.4/14.6
312
5.70
0.0257
6.23
77 ( 8.3
93 ( 9.3
P(NIPA-co-
94.5/5.5
306
5.27
0.0238
6.48
39 ( 4.7
4.8 ( 9.3
DMAEMA)-Si P(NIPA-co-DMAA)-Si
96.2/3.8
307
6.45
0.0291
5.86
40 ( 5.1
75 ( 6.1
P(NIPA-coVBTA)-Si
a
Determined by 1H NMR spectra of the corresponding free polymers. b Defined as the temperature where the optical transmittance of the aqueous solution of the corresponding free polymer was equal to 90%. c Determined by TG measurements. d Calculated using the molecular weight of PNIPA (Mp = 4.3 104). e Measured in water. f Standard deviation.
absence of residual chemicals in the supernatant solution was confirmed by ultravioletvisible light (UV-vis) spectra measured with a UV-vis spectrophotometer (Model V-550, JASCO, Tokyo, Japan). The washed silica particles were dried at 323 K for 12 h. The filtrate after polymerization was concentrated by evaporation and poured into a large volume of n-hexane to precipitate free polymer chains. The collected polymer was dissolved in water, and the solution was dialyzed against water using a cellulose dialysis membrane (MWCO 1000) for a few days. Finally, the polymer was obtained by freeze-drying. In the present study, the four polymer-grafted silica particles are abbreviated as PNIPA-Si, P(NIPA-co-VBTA)-Si, P(NIPA-coDMAEMA)-Si, and P(NIPA-co-DMAA)-Si. Similarly, the four free polymers are labeled as PNIPA, P(NIPA-co-VBTA), P(NIPA-co-DMAEMA), and P(NIPA-co-DMAA). 2.3. Characterization of Polymers. We carried out the following experiments using the free polymers obtained from the polymerization solutions as mentioned in the previous section and assumed that the grafted polymers on the silica surface have the similar properties as the corresponding free polymers. 1 H NMR spectra of the copolymers were recorded on a Model Lambda-500 spectrometer (JEOL, Tokyo, Japan) using D2O as the solvent to determine their monomer compositions. The peak areas used for the calculation were as follows: the peak areas at 1.2 ppm corresponding to the dimethyl protons of isopropyl groups in NIPA unit, at 3.1 ppm corresponding to the trimethyl protons of ammonium groups in VBTA unit, at 2.4 ppm corresponding to the dimethyl protons of amino groups in DMAEMA unit, and ∼3.0 ppm corresponding to the dimethyl protons adjacent to the N atom in DMAA unit. The phase transition of the polymers in water was investigated by optical transmittance measurements. The equilibrium values of optical transmittance of the polymer aqueous solution at different temperatures were obtained at 500 nm, using a UV-vis spectrophotometer, where the polymer concentration was 20 g/L. The temperature of the sample and the reference cells was controlled by a circulating water bath, and the temperature of water in the reference cell was monitored. The LCST was defined as the temperature where the optical transmittance of the solution was equal to 90%. The molecular weight of PNIPA (Mp) was determined by the relationship between the intrinsic viscosity in water and the molecular weight26,27 and found Mp = 4.3 104. For the sake of
convenience, we assumed that the copolymers in the present study have the same molecular weight, because the major component of each copolymer was NIPA, as will be described in section 3.1. 2.4. Characterization of Polymer-Grafted Silica Particles. To obtain the amount of polymers grafted onto the silica surface, thermogravimetric (TG) analysis was carried out using a Shimadzu Model DTG-60 instrument (Kyoto, Japan) from room temperature to 1173 K in air, using a heating rate of 10 K min1, where the sample temperature was kept constant at 393 and 1173 K for 20 and 10 min, respectively. The amount of grafted polymers was determined by the difference between the weight losses of the polymer-grafted silica and the MPTS-Si in the range of 3931173 K.28,29 The temperature dependence of the surface potential of the polymer-grafted silica particles was investigated in water at 298 and 313 K, using a zeta potential analyzer (ZEECOM, Microtec Co., Ltd., Chiba, Japan), because the adsorption experiments were performed at these temperatures, as will be described in section 2.5. The surface potential of the silica particle before polymer modification was also obtained for comparison. 2.5. Adsorption Experiments. All adsorption experiments were carried out in a batch system using a three-neck flask equipped with a stirrer and kept in a water bath at a specific temperature. Before starting the BSA adsorption experiment, the polymer-grafted silica particles were placed into 100 mL of water at 298 K, and the solution was stirred at 50 rpm. After 12 h, 100 mL of an aqueous solution containing 1000 ppm of BSA was added into the above solution to start the BSA adsorption experiment, where the initial concentration of BSA was 500 ppm, and the dosage of the polymer-grafted silica particles was determined so that the concentration of the grafted polymers was 75 ppm. The solution was stirred at 200 rpm during the adsorption experiment. The temperature-swing operation between 298 K and 313 K was conducted as follows: (i) the flask containing the solution was initially immersed in a water bath at 298 K; (ii) after 2 h, the flask was quickly transferred to another water bath at 313 K; (iii) after 2 h, the flask was brought back into the water bath at 298 K; (iv) procedures (ii) and (iii) were repeated. After the flask was transferred to the other water bath, the solution in the flask reached almost the same temperature as the water bath within 10 min. It should be noted that the denaturation temperature of BSA is ∼330 K,30 which is higher 12360
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Figure 4. Estimated size of the grafted polymers on a silica surface.
Figure 3. Optical transmittance of polymer aqueous solutions, as a function of temperature. The wavelength used was 500 nm.
than the temperatures applied in the adsorption experiments. The concentration of BSA in the aqueous solution was determined by UV absorbance at 278 nm measured by a UV-vis spectrophotometer after filtration through a cellulose acetate membrane filter (0.45 μm, Advantec, Dublin, CA): we checked the effect of the filtration on the BSA concentration using the BSA solutions without adsorbent and confirmed that the change of the BSA concentration is negligibly small. The amount of BSA adsorbed onto the polymer-grafted silica particles was calculated from the mass balance. In general, the pH value probably should be constant during the adsorption experiments, because the change in the solution pH leads to the change in the charge distribution of BSA and therefore influences its adsorption behavior. However, the coexisting solutes, especially ions also strongly affect the BSA adsorption behavior, resulting in the complication of the adsorption phenomenon. For this reason, we used not buffer solutions but water as the solvent. Before the adsorption experiments, we measured the pH values of the solutions under the same conditions as in the adsorption experiments using a pH meter (Argus, Sentron., Roden, The Netherlands) and found that the pH values at 298 and 313 K were 7.88.2 and 7.48.2, respectively. Considering that the isoelectric point of BSA is located at pH ∼4.8,31 the net charge of BSA is thought to be negative during the adsorption experiments in the present study.
P(NIPA-co-DMAEMA) and P(NIPA-co-DMAA) also showed a sharp change in the optical transmittance at 306307 K, whereas the optical transmittance of P(NIPA-co-VBTA) solution decreased gradually with increasing temperature. It is wellknown that the copolymerization of a hydrophilic monomer with NIPA results in an increase in the LCST.33 Especially, the gradual decrease in the transmittance of the P(NIPA-co-VBTA) solution is attributed to the relatively large fraction of the positively charged VBTA, which strongly prevents dehydration and coagulation of the copolymers. 3.3. Estimation of Grafted Polymer Size. The amounts of the grafted polymer estimated by the thermogravimetry (TG) measurements (denoted as w) are listed in Table 1. The amounts of the grafted copolymers were similar to each other, while that of the grafted PNIPA was almost twice as much as those of the copolymers grafted on the silica surface. The grafted polymer density on the silica surface (Fp) is thought to have a considerable effect on the adsorption and desorption behaviors of BSA. Using the amount of the grafted polymer (w) and the specific surface area of the silica particle (SBET = 3.1 m2/g), we estimated the values of Fp as well as the distance between the grafted polymers (d), under the following assumptions: (i) all grafted polymers have the same molecular weight as PNIPA, Mp = 4.3 104; (ii) the polymers are uniformly grafted on the silica surface. Based on these assumption, Fp and d are simply given by Fp ¼
sffiffiffiffiffi 1 d ¼ Fp
3. RESULTS AND DISCUSSION 3.1. Molar Compositions of Copolymers. The molar com1
positions of the copolymers determined by H NMR spectra are summarized in Table 1. The mole fraction of VBTA units in P(NIPA-co-VBTA) was relatively larger than that in the feed solution (5 mol %), while the other two copolymers had compositions similar to the feed solutions. This indicates that VBTA has high reactivity, compared to other monomers. In all copolymers, however, NIPA is a major component, which suggests that these copolymers would exhibit more or less temperature responsiveness. 3.2. LCST of Polymers. The optical transmittance of the polymer aqueous solutions are shown in Figure 3, as a function of temperature, and each LCST (which is defined as the temperature at 90% transmittance) is listed in Table 1. PNIPA showed a sharp change in the optical transmittance at ∼306 K: this LCST is in good agreement with the reported value.32
wNA Mp SBET
ð1Þ
ð2Þ
where NA is Avogadro’s number. The resultant values are summarized in Table 1. Similar values for Fp and d have been reported by other research groups, using almost the same procedure to graft the polymers on the silica surface.29,34 In addition to the values of Fp and d, the chain dimension of the grafted polymer is also an important factor to consider the adsorption of BSA. As a measure of the polymer chain dimension on the silica surface, the radius of gyration of PNIPA in water (Rg) was employed. From the molecular weight, Rg can be obtained by Rg ¼ KMp a
ð3Þ
where K = 0.0224 nm and a = 0.54 at 298 K.27 The calculated value with Mp = 4.3 104 is Rg = 7.1 nm. As for P(NIPA-coVBTA)-Si and P(NIPA-co-DMAEMA)-Si, the values of Rg must be much larger than Rg = 7.1 nm because their grafted copolymers are positively charged. From the values of d and Rg, the structures 12361
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Figure 6. Amounts of BSA adsorbed onto the copolymer-grafted silica particles at 298 K (q298) and 313 K (q313), and the difference between them (Δq = q298 q313). P(N-V)-Si and P(N-D)-Si represent P(NIPAco-VBTA)-Si and P(NIPA-co-DMAEMA)-Si, respectively.
Figure 5. Time and temperature dependences of the amount of BSA adsorbed onto the polymer-grafted silica particles: (a) PNIPA-Si, (b) P(NIPA-co-VBTA)-Si, (c) P(NIPA-co-DMAEMA)-Si, and (d) P(NIPA-co-DMAA)-Si.
of the grafted polymers on the silica surface can be illustrated as shown in Figure 4. 3.4. Surface Potential of Polymer-Grafted Silica Particle. The surface potentials of the polymer-grafted silica particles in water at 298 and 313 K (ψ) are summarized in Table 1, where the surface potential of the silica particle before polymer modification is also listed for comparison. The surface potential of the silica particle was negative, because of the deprotonation of silanol groups on the silica surface, and slightly increased with increasing temperature. As for the silica particles grafted with the neutral polymers—that is, PNIPA-Si and P(NIPA-co-DMAA)Si—the values of ψ were also negative and approximately doubled with increasing temperature from 298 K to 313 K. This may be because the conformations of PNIPA and P(NIPA-coDMAA) on the silica surface change from expanded coil to compact globule above the LCST at ∼306 K, and the influence of the silica surface becomes more influential. As for P(NIPA-coVBTA)-Si, the values of ψ at 298 K and 313 K were positive, because VBTA is a quaternary ammonium salt and positively charged in water. As for P(NIPA-co-DMAEMA)-Si, on the other
hand, the value of ψ at 298 K was positive, whereas that at 313 K was almost zero. In aqueous solutions, some of DMAEMA groups in P(NIPA-co-DMAEMA)-Si can be protonated, because DMAEMA is a tertiary amine with pKa = 8.4.3537 With increasing temperature across the LCST at ∼306 K, however, the copolymer that is grafted on the silica surface dehydrates and undergoes a sharp transition from hydrophilic coil to hydrophobic globule (see Figure 3). This dehydration of the grafted copolymer leads to the deprotonation of DMAEMA groups and, thus, also to a near-zero value of ψ at 313 K. In the temperatureswing adsorption of BSA, the negatively charged BSA would be adsorbed by the positively charged copolymers on the silica surface through the electrostatic attraction, and then the electrostatic attraction between the adsorbed BSA and the grafted polymers should be cut off by the temperature change to desorb BSA. Based on this concept, P(NIPA-co-DMAEMA)-Si is considered to be promising for temperature-swing adsorption, because its surface potential changes from positive to almost zero as the temperature increases from 298 K to 313 K. 3.5. Temperature-Swing Adsorption of BSA. The time and temperature dependences of the adsorption amounts of BSA onto the polymer-grafted silica particles (q) are shown in Figure 5: for the sake of convenience, the temperature of the water bath is used as a substitute for the sample temperature (see also section 2.5). Both PNIPA-Si and P(NIPA-co-DMAA)-Si hardly adsorbed BSA, because these polymer-grafted silica particles, as well as BSA, are negatively charged, as mentioned in section 3.4. On the other hand, positively charged P(NIPA-coVBTA)-Si adsorbed BSA at 298 K through the electrostatic attraction between them, and desorbed some of the BSA that was adsorbed at 298 K after a temperature increase to 313 K. Note that this adsorption and desorption behavior can be repeated by the temperature-swing operation. The BSA desorption from P(NIPA-co-VBTA)-Si may result from the decrease in the number of VBTA groups accessible to BSA, because of the coil-to-globule transition of the grafted copolymers with increasing temperature. Similarly, P(NIPA-co-DMAEMA)-Si repeatedly adsorbed BSA at 298 K and desorbed some of the preadsorbed BSA at 313 K. To assess the adsorption and desorption amounts of BSA for P(NIPA-co-VBTA)-Si and P(NIPA-co-DMAEMA)-Si shown in Figure 5, the average adsorption and desorption amounts were calculated as follows: (i) the average of the adsorption amounts at times of t = 2, 6, and 10 h was defined as the average adsorption 12362
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Table 2. Adsorption States of BSA on Copolymer-Grafted Silica Particles adsorption density of BSA, FBSA [molecules/nm2] sample name
copolymer chains per adsorbed BSA, Np [chains/molecule]
at 298 K
at 313 K
at 298 K
at 313 K
P(NIPA-co-VBTA)-Si
0.0170 ( 0.0009
0.0146 ( 0.0002
1.52 ( 0.08
1.77 ( 0.03
P(NIPA-co-DMAEMA)-Si
0.0123 ( 0.0005
0.0098 ( 0.0010
1.94 ( 0.08
2.42 ( 0.28
the sizes of the grafted copolymer (see section 3.3 and Figure 4) and BSA (4 4 11.5 nm),38 the adsorption and desorption behavior of BSA on and from P(NIPA-co-DMAEMA)-Si is considered as shown in Figure 7. At 298 K, a single BSA molecule is adsorbed by a few copolymers on the silica surface. The copolymers “wrapping” the BSA molecule are unlikely to dehydrate and deprotonate, even after the temperature increases to 313 K (above LCST), because of the hydrophilicity of BSA. Accordingly, most of the adsorbed BSA molecules are difficult to be desorbed, while only a small number of them, which may be adsorbed at the outer surface of the grafted copolymer brush, can be released as illustrated in Figure 7. Judging from the values of FBSA and Np, the BSA adsorption and desorption behavior for P(NIPA-co-VBTA)-Si is thought to be similar to that for P(NIPA-co-DMAEMA)-Si. Figure 7. Possible illustration of the adsorption and desorption behavior of BSA on and from P(NIPA-co-DMAEMA)-Si by the temperatureswing operation between 298 K and 313 K.
amount at 298 K (denoted as q298); (ii) similarly, the average value of the adsorption amounts at times of t = 4, 8, and 12 h represents the average adsorption amount at 313 K (denoted as q313); (iii) the difference between q298 and q313 was defined as the average desorption amount (Δq q298 q313). The resultant values are shown in Figure 6. The adsorption amounts of BSA onto P(NIPA-co-DMAEMA)-Si was smaller than that onto P(NIPA-co-VBTA)-Si, whereas the BSA desorption amounts from them are similar to each other. As mentioned in section 3.4, we had expected that P(NIPA-co-DMAEMA)-Si would adsorb BSA at 298 K and desorb most of the preadsorbed BSA at 313 K, because the surface potential of P(NIPA-coDMAEMA)-Si changes from ∼40 mV to almost zero as the temperature increases from 298 to 313 K (see Table 1). To consider the reason for this unexpected result, we estimated the adsorption and desorption states of BSA on P(NIPA-coDMAEMA)-Si. The adsorption density of BSA on the copolymer-grafted silica surface (FBSA) can be calculated as follows: FBSA ¼
qwNA MBSA SBET
ð4Þ
where MBSA is the molecular weight of BSA (MBSA = 6.75 104).38 From eqs 1 and 4, the number of copolymers per adsorbed BSA (Np) is given by ! Fp 1 MBSA Np ¼ ¼ ð5Þ q Mp FBSA The calculated values of FBSA and Np for P(NIPA-coDMAEMA)-Si are listed in Table 2: the values for P(NIPA-coVBTA)-Si are also computed. The value of Np at 298 K for P(NIPA-co-DMAEMA)-Si indicates that the ratio of copolymer chains to adsorbed BSA was ∼2:1. Based on this value, as well as
4. CONCLUSIONS We prepared silica particles grafted with poly(N-isopropylacrylamide) (PNIPA) copolymers as an adsorbent for the temperature-swing adsorption of bovine serum albumin (BSA) in water, where vinylbenzyl trimethylammonium chloride (VBTA), 2-(dimethylamino)ethyl methacrylate (DMAEMA), or N,Ndimethylacrylamide (DMAA) was employed as a comonomer. We examined the characteristics of the polymer-grafted silica particles, as well as the corresponding free polymers, and then applied those silica particles to the temperature-swing adsorption experiments. As a result, we have drawn the following conclusions: (1) P(NIPA-co-DMAEMA) and P(NIPA-co-DMAA) show a sharp phase transition at temperatures near the LCST of PNIPA, whereas P(NIPA-co-VBTA) gradually changes from coil to globule with increasing temperature, because of the strong repulsion between the positively charged VBTA groups. (2) The surface potential of P(NIPA-co-VBTA)-Si is positive at both 298 and 313 K, whereas that of P(NIPA-coDMAEMA)-Si changes from positive to almost zero as the temperature increases from 298 K to 313 K. (3) P(NIPA-co-VBTA)-Si and P(NIPA-co-DMAEMA)-Si adsorb BSA in water while PNIPA-Si and P(NIPA-coDMAA)-Si fail to adsorb BSA, indicating that BSA is adsorbed by the electrostatic attraction between the negatively charged BSA and the positively charged copolymers on the silica surface. (4) P(NIPA-co-VBTA)-Si and P(NIPA-co-DMAEMA)-Si can repeatedly adsorb BSA at 298 K and desorb some of the preadsorbed BSA at 313 K via the temperatureswing operation. Although we succeeded in the temperature-swing adsorption of BSA using the silica particles grafted with temperatureresponsive copolymers, the desorption amounts of BSA were rather small. This inefficiency might be improved by using the short polymer brush with high density, which can be prepared by 12363
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’ AUTHOR INFORMATION Corresponding Author
*Tel.: +81-45-924-5419. Fax: +81-45-924-5419. E-mail: smorisada@ chemenv.titech.ac.jp.
’ ACKNOWLEDGMENT The authors thank Kohjin Co., Ltd., and Fuji Silycia Chemical, Ltd., for the generous gifts of two types of acrylamide monomers (NIPA and DMAA) and silica particles, respectively. 1H NMR spectra were measured at Center for Advanced Materials Analysis (Suzukakedai), Technical Department, Tokyo Institute of Technology. This work was partly supported by the Grants-inAid for Scientific Research (No. 20760512) from the Ministry of Education, Culture, Sports, Science and Technology in Japan (MEXT) and by the Core-to-Core Program (No. 18004) promoted by Japan Society for the Promotion of Science (JSPS). ’ REFERENCES (1) Heskins, M.; Guillet, J. E. Solution Properties of Poly(Nisopropylacrylamide). J. Macromol. Sci., Chem. 1968, 2 (8), 1441–1455. (2) Cammas, S.; Suzuki, K.; Sone, C.; Sakurai, Y.; Kataoka, K.; Okano, T. Thermo-Responsive Polymer Nanoparticles with a Core Shell Micelle Structure as Site-Specific Drug Carriers. J. Controlled Release 1997, 48 (23), 157–164. (3) Kono, K.; Nakai, R.; Morimoto, K.; Takagishi, T. Thermosensitive Polymer-Modified Liposomes That Release Contents around Physiological Temperature. Biochim. Biophys. Acta 1999, 1416 (12), 239–250. (4) Kikuchi, A.; Okano, T. Pulsatile Drug Release Control Using Hydrogels. Adv. Drug Delivery Rev. 2002, 54 (1), 53–77. (5) Kwon, O. H.; Kikuchi, A.; Yamato, M.; Sakurai, Y.; Okano, T. Rapid Cell Sheet Detachment from Poly(N-isopropylacrylamide)Grafted Porous Cell Culture Membranes. J. Biomed. Mater. Res. 2000, 50 (1), 82–89. (6) Yamato, M.; Kwon, O. H.; Hirose, M.; Kikuchi, A.; Okano, T. Novel Patterned Cell Coculture Utilizing Thermally Responsive Grafted Polymer Surfaces. J. Biomed. Mater. Res. 2001, 55 (1), 137–140. (7) Nagase, K.; Kobayashi, J.; Okano, T. Temperature-Responsive Intelligent Interfaces for Biomolecular Separation and Cell Sheet Engineering. J. R. Soc. Interface 2009, 6, S293–S309. (8) Ohashi, K.; Yokoyama, T.; Yamato, M.; Kuge, H.; Kanehiro, H.; Tsutsumi, M.; Amanuma, T.; Iwata, H.; Yang, J.; Okano, T.; Nakajima, Y. Engineering Functional Two- and Three-Dimensional Liver Systems in vivo Using Hepatic Tissue Sheets. Nat. Med. 2007, 13 (7), 880–885. (9) Teal, H. E.; Hu, Z. B.; Root, D. D. Native Purification of Biomolecules with Temperature-Mediated Hydrophobic Modulation Liquid Chromatography. Anal. Biochem. 2000, 283 (2), 159–165. (10) Kawaguchi, H.; Fujimoto, K.; Mizuhara, Y. Hydrogel Microspheres III. Temperature-Dependent Adsorption of Proteins on Poly(N-isopropylacrylamide) Hydrogel Microspheres. Colloid Polym. Sci. 1992, 270 (1), 53–57. (11) Norde, W. Adsorption of Proteins from Solution at the SolidLiquid Interface. Adv. Colloid Interface Sci. 1986, 25 (4), 267– 340. (12) Kleijn, M.; Norde, W. The Adsorption of Proteins from Aqueous Solution on Solid Surfaces. Heterog. Chem. Rev. 1995, 2 (3), 157–172.
ARTICLE
(13) Norde, W. My Voyage of Discovery to Proteins in Flatland and Beyond. Colloid Surf. B 2008, 61 (1), 1–9. (14) Kim, J.; Qian, W.; Al-Saigh, Z. Y. Measurements of Water Sorption Enthalpy on Polymer Surfaces and Its Effect on Protein Adsorption. Surf. Sci. 2011, 605 (34), 419–423. (15) Ela€issari, A.; Holt, L.; Meunier, F.; Voisset, C.; Pichot, C.; Mandrand, B.; Mabilat, C. Hydrophilic and Cationic Latex Particles for the Specific Extraction of Nucleic Acids. J. Biomater. Sci. Polym. Ed. 1999, 10 (4), 403–420. (16) Ela€issari, A.; Bourrel, V. Thermosensitive Magnetic Latex Particles for Controlling Protein Adsorption and Desorption. J. Magn. Magn. Mater. 2001, 225 (12), 151–155. (17) Moselhy, J.; Wu, X. Y.; Nicholov, R.; Kodaria, K. In vitro Studies of the Interaction of Poly(NIPAm/MAA) Nanoparticles with Proteins and Cells. J. Biomater. Sci., Polym. Ed. 2000, 11 (2), 123–147. (18) Gan, D.; Lyon, L. A. Synthesis and Protein Adsorption Resistance of PEG-Modified Poly(N-isopropylacrylamide) Core/Shell Microgels. Macromolecules 2002, 35 (26), 9634–9639. (19) Nolan, C. M.; Reyes, C. D.; Debord, J. D.; Garc€ia, A. J.; Lyon, L. A. Phase Transition Behavior, Protein Adsorption, and Cell Adhesion Resistance of Poly(ethylene glycol) Cross-Linked Microgel Particles. Biomacromolecules 2005, 6 (4), 2032–2039. (20) Grabstain, V.; Bianco-Peled, H. Mechanisms Controlling the Temperature-Dependent Binding of Proteins to Poly(N-isopropylacrylamide) Microgels. Biotechnol. Prog. 2003, 19 (6), 1728–1733. (21) Silva, C. S. O.; Baptista, R. P.; Santos, A. M.; Martinho, J. M. G.; ^ . Adsorption of Human IgG on to Poly(NCabral, J. M. S.; Taipa, M. A isopropylacrylamide)-Based Polymer Particles. Biotechnol. Lett. 2006, 28 (24), 2019–2025. (22) Eke, I.; Elmas, B.; Tuncel, M.; Tuncel, A.; New, A Highly Stable Cationic-Thermosensitive Microgel: Uniform IsopropylacrylamideDimethylaminopropylmethacrylamide Copolymer Particles. Colloids Surf., A 2006, 279 (13), 247–253. (23) Huo, D.; Li, Y.; Qian, Q.; Kobayashi, T. TemperaturepH Sensitivity of Bovine Serum Albumin Protein-Microgels Based on CrossLinked Poly(N-Isopropylacrylamide-co-Acrylic Acid). Colloids Surf., B 2006, 50 (1), 36–42. (24) Morisada, S.; Namazuda, K.; Kanda, H.; Hirokawa, Y.; Nakano, Y. Temperature-Swing Adsorption of Proteins in Water Using NIsopropylacrylamide Copolymer Gel Particles. Adv. Powder Technol. 2010, 21 (1), 28–33. (25) Philipse, A. P.; Vrij, A. Preparation and Properties of Nonaqueous Model Dispersions of Chemically Modified, Charged Silica Spheres. J. Colloid Interface Sci. 1989, 128 (1), 121–136. (26) Fujishige, S. Intrinsic ViscosityMolecular Weight Relationships for Poly(N-isopropylacrylamide) Solutions. Polym. J. 1987, 19 (3), 297–300. (27) Kubota, K.; Fujishige, S.; Ando, I. Solution Properties of Poly(N-isopropylacrylamide) in Water. Polym. J. 1990, 22 (1), 15–20. (28) Browne, T.; Chaimberg, M.; Cohen, Y. Graft Polymerization of Vinyl Acetate onto Silica. J. Appl. Polym. Sci. 1992, 44 (4), 671–677. (29) Nguyen, V.; Yoshida, W.; Cohen, Y. Graft Polymerization of Vinyl Acetate onto Silica. J. Appl. Polym. Sci. 2003, 87 (2), 300–310. (30) Poole, S.; West, S. I.; Fry, J. C. Effects of Basic Proteins on the Denaturation and Heat-Gelation of Acidic Proteins. Food Hydrocolloids 1987, 1 (4), 301–316. (31) Tanford, C.; Swanson, S. A.; Shore, W. S. Hydrogen Ion Equilibria of Bovine Serum Albumin. J. Am. Chem. Soc. 1955, 77 (24), 6414–6421. (32) Schild, H. G. Poly(N-isopropylacrylamide): Experiment, Theory and Application. Prog. Polym. Sci. 1992, 17 (2), 163–249. (33) Rzaev, Z. M. O.; Dincer, S.; Piskin, E. Functional Copolymers of N-Isopropylacrylamide for Bioengineering Applications. Prog. Polym. Sci. 2007, 32 (5), 534–595. (34) Chaimberg, M.; Cohen, Y. Free-Radical Graft-Polymerization of Vinylpyrrolidone onto Silica. Ind. Eng. Chem. Res. 1991, 30 (12), 2534–2542. (35) Hoogeveen, N. G.; Stuart, M. A. C.; Fleer, G. J.; Frank, W.; Arnold, M. Novel Water-Soluble Block Copolymers of Dimethylaminoethyl 12364
dx.doi.org/10.1021/ie200779w |Ind. Eng. Chem. Res. 2011, 50, 12358–12365
Industrial & Engineering Chemistry Research
ARTICLE
Methacrylate and Dihydroxypropyl Methacrylate. Macromol. Chem. Phys. 1996, 197 (8), 2553–2564. (36) van de Wetering, P.; Zuidam, N. J.; van Steenbergen, M. J.; van der Houwen, O.; Underberg, W. J. M.; Hennink, W. E. A Mechanistic Study of the Hydrolytic Stability of Poly(2-(dimethylamino)ethyl methacrylate). Macromolecules 1998, 31 (23), 8063–8068. (37) B€ut€un, V.; Armes, S. P.; Billingham, N. C. Synthesis and Aqueous Solution Properties of Near-Monodisperse Tertiary Amine Methacrylate Homopolymers and Diblock Copolymers. Polymer 2001, 42 (14), 5993–6008. (38) Baszkin, A.; Lyman, D. J. The Interaction of Plasma Proteins with Polymers. I. Relationship between Polymer Surface Energy and Protein Adsorption/Desorption. J. Biomed. Mater. Res. 1980, 14 (4), 393–403.
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