Novel Membrane Adsorbers with Grafted Zwitterionic Polymers

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Novel Membrane Adsorbers with Grafted Zwitterionic Polymers Synthesized by Surface-Initiated ATRP and Their Salt-Modulated Permeability and Protein Binding Properties Qian Yang and Mathias Ulbricht* Lehrstuhl für Technische Chemie II, Universität Duisburg-Essen, 45117 Essen, Germany S Supporting Information *

ABSTRACT: A novel zwitterionic polymer functionalized porous membrane adsorber was obtained by grafting poly(N,Ndimethyl-N-methacryloyloxyethyl-N-(3-sulfopropyl) ammonium betaine) (polySPE) to poly(ethylene terephthalate) (PET) track-etched membrane surface via surface-initiated atom transfer radical polymerization (SI-ATRP). The ATRP conditions were optimized, the thus established grafting was well-controlled, and the degree of grafting could be adjusted. Functionalized membranes with a degree of grafting of about 3.5 μg/cm2 relative to the specific surface area showed almost zero values of zeta potential estimated from the trans-membrane streaming potential measurements. Typical “anti-polyelectrolyte” effect was observed for the polySPE grafted membranes. Flux through the membrane was reduced by adding chaotropic chloride and perchlorate salts to the solution which extended the polySPE chains grafted on the membrane pore wall. Perchlorate salt exhibited much stronger effect on polySPE chain conformation than chloride salt and for a membrane with a degree of grafting of 2.7 μg/cm2, even 2 mM KClO4 could extend the thickness of the polymer layer to more than two times (∼43 nm) of that in pure water (∼20 nm). On the contrary, small amounts of kosmotropic ions (10 mM SO42‑) further “salted out” the polySPE chains and led to a slightly increased flux. PolySPE grafted PET membranes with different degree of grafting were then used as membrane adsorber for protein binding. Human IgG was used as model protein and the binding capacity was evaluated under both static (no convective flow through the membrane) and dynamic conditions (flow-through conditions). Static adsorption experiments showed that IgG could be loaded to the membrane at medium salt concentration and 85−95% of bound protein could be eluted at either low (zero) or very high salt concentrations. Dynamic flow-through experiments then revealed the influences of salt concentration and salt type on IgG binding. Effects of two chaotropic salts, NaCl and NaClO4, were evaluated. Slight but not negligible binding of IgG from pure water was suppressed by adding NaCl. IgG binding was then increased in the NaCl concentration range of 100−500 mM and reached a maximum binding capacity value at about 500 mM. Further increase of NaCl concentration led to a decreased binding again. KClO4 showed similar effects onto IgG binding, but this salt functions in a much lower and much narrower concentration range. All results with respect to grafted layer swelling and protein binding followed the empirical Hofmeister series. KEYWORDS: zwitterionic polymer, ATRP, track-etched membrane, protein binding laboratories and industries.1−4 Membrane chromatography is very promising and has very good characteristics for bioseparation, especially for the isolation and purification of proteins.5−8 These tasks are traditionally performed by using

1. INTRODUCTION Chromatography is a technique with more than 100 years history and has been widely applied for both preparative and analytical purposes. It is by far the most important and mostly used technique for separation and purification of proteins. As a newly developed variant, membrane-adsorber-based chromatography processes have been intensively investigated in recent years and have been proven to be a robust technique in © 2012 American Chemical Society

Received: April 10, 2012 Revised: July 21, 2012 Published: July 23, 2012 2943

dx.doi.org/10.1021/cm301116p | Chem. Mater. 2012, 24, 2943−2951

Chemistry of Materials

Article

Scheme 1. Schematic Representation for (a) the Grafting of PolySPE on Track-Etched PET Membranes and (b) the Extension of polySPE Chains Grafted on the Cylindrical Pore Wall of PET Membranes by Increase of Salt Concentration

column chromatography processes with packed beds and/or gels; that has several major drawbacks.2 The intrinsic pressure drop across the packed beds is always very high in conventional chromatography processes. Also, for packed bed chromatography processes the diffusion of solute species to the binding sites in the solid phase materials, especially for macromolecules or particles like proteins or viruses, is very slow. This slow diffusion then leads to a longer processing time and lower process throughput and causes poor utilization of binding sites. Furthermore, the interparticle volume within the packed bed increases the required volume of eluent solution and further increases dilution of the product.9 In contrast, in membrane chromatography, binding sites are along the path of the feed flow through the membrane pores and target molecules and particles can access the binding sites on the membrane (pore) surface directly by bulk convection.10 Consequently, the total mass transfer resistance of the membrane is much lower and membrane stacks have not such high pressure drop compared with traditional packed bed columns. Furthermore, the membrane chromatography is much easier to fit in scale-up industrial devices.11−13 Membrane adsorbers used in membrane chromatography are generally obtained by grafting functional polymers onto existing membranes; typical techniques for membrane surface modification are well-demonstrated in several recent reviews despite that the purposes of modification are diverse.14,15 Among different membrane chromatography categories, ionexchange membrane adsorber based process is the mostly investigated and applied method for biomolecule separation and purification. It functions based on electrostatic force, which is typically endowed by functional polymers with charged groups such as carboxylic acid, sulfonic acid, diethylaminoethyl and quaternary ammonium ethyl, and these charged groups bind to the target molecules or particles, e.g., proteins or viruses. However, this process involves the reversible

adsorption of the proteins onto the membrane adsorbers by the electrostatic attraction between the ionic groups on the membrane surface and the protein which imposes the risk of denaturing the protein. Compared with polymers bearing “conventional” charged groups as mentioned above, sulfobetaine based zwitterionic polymers, which display both positive and negative charges in every single repeating unit, provide the possibility of maintaining the native protein conformation throughout the adsorption by engaging a “soft” ionic interaction with proteins.16,17 With “conventional” ion-exchangers, a salt gradient elution solution with very high salt concentration will be needed to recover the protein from the adsorber. Therefore, the target proteins are always obtained in a high salt buffer and further steps have to be taken to remove the salts which could be costly and time-consuming. Zwitterionic polymers give a chance to realize protein chromatography with pure water or dilute aqueous solutions of electrolytes as eluents, and this has led to the new zwitterionic/hydrophilic interaction liquid chromatography (ZIC-HILC).18−23 Viklund et al. had synthesized macroporous polymeric monoliths from N,Ndimethyl-N-methacryloyloxyethyl-N-(3-sulfopropyl) ammonium betaine (SPE) and used them as stationary phase for chromatography. Benefiting from the outstanding characteristics of zwitterionic materials, fast separations of basic proteins and peptides were achieved under mild conditions with low salt elution solutions.17 Similar work from the same group revealed that the interaction between the zwitterionic polymers and proteins could be efficiently controlled over a broad range by addition of low concentrations of chaotropic ions.16 In this study, a novel porous membrane adsorber with grafted zwitterionic polymer was prepared by growing polySPE in poly(ethylene terephthalate) track-etched membrane (PET TEM) via surface-initiated ATRP (Scheme 1). The grafting was well-controlled by adjusting the ATRP conditions and a linear increase of degree of grafting (DG) was achieved in the first 4 h 2944



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(Porous Materials, Inc., Ithaca, NY) was used, measuring the membrane first in the dry state (nitrogen flow as function of pressure), then in the wet state (pores filled with 1,1,2,3,3,3hexafluoropropene; “Galwick”, PMI; surface tension 16 dyn cm−1), and thereafter calculating the average pore radius. Then, the hydrodynamic thickness of the grafted layer on the pore wall (HLT) was obtained as the difference between the hydrodynamic pore radii of the unmodified and of the grafted membrane. 2.3. Surface-Initiated ATRP of SPE on PET Membrane. 2.3.1. Initiator Immobilization. Immobilization of the ATRP initiator onto the PET TEM was achieved by the reaction between BMPB and hydroxyl groups on the PET surface. The membrane samples were put in a vessel with 10 mL freshly dried acetonitrile containing DMAP (5 mM) and TEA (10 mM), and 100 μL BMPB. The vessel was then sealed and put on a shaker. After reaction for 2 h at room temperature, membranes were taken out and rinsed with acetonitrile, water and ethanol, and then dried in a vacuum oven at 40 °C overnight. 2.3.2. ATRP of SPE from PET Membrane. Initiator immobilized membrane samples were put in Schlenck flask equipped with rubber stoppers and the flask was sealed. Then, the flask was evacuated and backfilled with argon three times. Bpy was dissolved in water/ methanol mixture solution (1:1, v/v) and then purged with nitrogen for 30 min. Thereafter, copper(I) bromide was added to the solution under strong stirring and argon stream. In some cases, certain amount of copper(II) bromide was also added to the system. After nitrogen purging for another 10 min, monomer SPE was added. Subsequently, reaction solution was cannulated into the flask (7 mL for each sample) and the reaction mixture was incubated at room temperature for predetermined reaction time. After ATRP reaction, a quenching solution was used to stop the polymerization: The membranes were quickly removed from the Schlenck flask and immersed in 50 mL 1:1 (v/v) methanol/water solution of 250 mg copper(II) bromide and 625 μL PMDETA. A water−methanol−ethanol washing sequence was then used to clean the membranes. After drying in vacuum oven at 40 °C overnight, the degree of grafting, DG (μg/cm2), was calculated by following equation:

reaction time. The swelling properties of the grafted polySPE chains were determined by monitoring the fluxes of different salt solutions through the membrane. Thus, the influences of salt type and salt concentration on the chain conformation of polySPE were investigated. IgG binding capacity of this membrane adsorber was evaluated under both static (no convective flow through the membrane) and dynamic conditions (flow-through conditions). Comprehensive studies on the effect of typical chaotropic ions (Cl− and ClO4−) on the IgG binding were then carried out and the effects of different salts were demonstrated.

2. EXPERIMENTAL SECTION 2.1. Materials. PET TEM with nominal pore diameter of 400 nm and a thickness of 23 μm were purchased from Oxyphen GmbH (Dresden, Germany). All membrane samples used in this study were cut from large sheets into circular specimen with a diameter of 25 mm. SPE was purchased from Aldrich and used as received. Acetonitrile was purified by refluxing with boric anhydride and distillation before use. Copper(I) bromide (Aldrich, 99.999%) and copper(II) bromide (Acros, 99+%) were used without further purification. 2-Bromo-2methylpropionyl bromide (BMPB, Aldrich), triethylamine (TEA, Aldrich), 2,2′-bipyridine (Bpy, Aldrich), 4-(N′,N′-dimethylamino) pyridine (DMAP, Fluka), N,N,N′,N″,N″-pentamethyl diethylenetriamine (PMDETA, Fluka) and methanol were also used as received. IgG (human serum) was from Sigma. BCA protein assay (Thermo Scientific, USA) was used for protein concentration determination. The water used in all syntheses and measurements was from a Milli-Q system (Millipore, USA). 2.2. Characterizations. 2.2.1. Zeta Potential from Streaming Potential. Zeta potentials of unmodified and polySPE grafted membranes were measured by a SurPASS electrokinetic analyzer (Anton-Paar GmbH, Austria). A cylindrical cell was used and the measurements were always started at pH 10 in a 1 × 10−3 M KCl solution prepared with Milli-Q water; the other pH values were adjusted by the addition of dilute HCl solutions. The streaming current has been measured and converted to the zeta potential using the Helmholtz-Smoluchowski model. All zeta potential data collected were averages of four measurements at the same pH. 2.2.2. Permeability and Hydrodynamic Layer Thickness. Measurements of pure water flux and fluxes of different salt solutions in thermostatted Amicon cells (Millipore, MA, U.S.A.) as well as calculations of reduction of pore diameter (for modified membranes) relative to the isoporous precursor membranes were done according to the earlier described procedures.24 To ensure that grafted zwitterionic polymer layers were completely adapted to corresponding surroundings, the membrane samples were equilibrated in water or salt solutions for at least 30 min before starting the experiments. Flux was measured by weighing the water permeates through the membrane in every 5 min and six continuous measurements were carried out to obtain the value for one sample. Typically, at least three parallel samples with similar DG were measured. The reported average flux values and standard deviations were calculated from the data for the parallel membrane samples. Permeability resulted from dividing the flux by the transmembrane pressure, and the pore size of the membrane was calculated from the permeability data by using the Hagen−Poiseuille equation

DG =

W1 − W0 Am

where W0 is the mass of the unmodified membrane and W1 is the mass of the membrane after modification and drying. Am represents the specific surface area of the membrane (194.3 cm2 for the samples with 4.9 cm2 geometric area used in this study; from BET data measured with the porometer SA 3100, Beckman-Coulter). The membrane samples were weighed using the balance GENIUS (accuracy: ± 10 μg) from Sartorius (Germany). 2.4. IgG Binding Property of PolySPE-Grafted PET Membrane. All binding experiments, in both static adsorption mode and dynamic flow-through mode, were performed by using 0.1 g/L IgG dissolved in different solvents including pure water, phosphate buffer solution (10 mM, pH 7.4) with or without NaCl and aqueous solutions of NaCl or KClO4. For each experiment, the membrane was prewetted before and washed after the adsorption step with the corresponding solvent used to dissolve the IgG. 2.4.1. Static Adsorption Mode. In a typical static adsorption mode experiment, membrane samples were immersed in the solvent for 30 min to prewet. Then each sample was wiped with filter paper and put into a vessel containing 6 mL IgG solution. After incubation at 25 °C for 24 h with slight shaking, membrane samples were taken out and the amount of bound IgG was determined by BCA protein assay and measuring spectrophotometrically the difference between the concentrations of IgG in the solution before and after contact with the membranes. The membrane after adsorption was first rinsed with 2 mL of corresponding solvent each for 6 times. For elution of IgG from the membrane, the sample was then wiped with filter paper and put into 6 mL desorption solution for 2 h. Finally, IgG concentration in desorption solution was measured by BCA assay as described above for binding experiments. For different solvents used in this study, calibration curves obtained from standard IgG solutions in corresponding solvents were used. The BCA protein assay with the

π Δpr 4 V = Δt 8ηL where V is the volume of the permeate relating to a single cylindrical membrane pore, Δt is the time interval, Δp is the transmembrane pressure, r is the pore radius, η is the viscosity of water, and L is the capillary length (i.e., the membrane thickness). This equation is valid assuming a cylindrical geometry of the membrane pores and equal size of all pores. First, pore radius and pore density of the unmodified membrane were calculated from gas flow/pore dewetting permporometry and the water permeability. The PMI capillary flow porometer 2945



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typical microplate procedure described in the instruction book has been applied using the Microplate Reader μQuant (Bio-Tek Instruments Inc., USA). The specific surface area of 194.3 cm2 (for the 25 mm diameter membrane sample; cf. Section 2.3.2) was used to calculate the protein binding capacity. 2.4.2. Dynamic Flow-through Mode. In a typical dynamic flowthrough mode experiment, membrane samples were first immersed in the solvent for 30 min to prewet. Then the membrane was sealed in a membrane holder (SWINNEX-25, Millipore, USA) and the holder was connected with a syringe containing 10 mL IgG solution. The syringe was driven by a syringe pump and the protein solution permeated through the membrane with a constant flow rate (0.1 mL/ min). The permeated protein solution was collected and the protein concentration was measured by BCA protein assay as described above for static adsorption (cf. Section 2.4.1).

3. RESULTS AND DISCUSSION 3.1. Surface-Initiated ATRP of SPE on PET Membrane. The reaction sequence for the grafting of polySPE to the membrane surface, including immobilization of the ATRP initiator and graft copolymerization, is summarized in Scheme 1a. The initiator was immobilized to the hydroxyl groups on the membrane surface directly and lead to a low initiator density. In some of our previous studies,10,24−26 we had achieved high grafted polymer chain density by first increasing hydroxyl group content on the membrane surface for initiator immobilization via a pretreatment step. Without such pretreatment step, hydroxyl density was 0.2 nm−2.26 The initiator immobilization conditions had been developed and proven to proceed at high conversion on the one hand; most important, the cylindrical pore morphology with very narrow size distribution of the PET TEM remained unchanged on the other hand.26 The pore diameter of the PET TEM was determined by permporometry, the average value for the batch used in this study was ∼690 nm. The membranes had a narrow pore size distribution (see Figure S1 in the Supporting Information) while the pore diameter was much higher than the value provided by the manufacturer. However, this was similar to previous studies and the possible reason, overetching during production, had been discussed before.25−27 Then, the graft copolymerization of SPE was initiated from PET membrane surface and the ATRP conditions were optimized. A 1:1 (v/v) water/methanol mixture solution was used as solvent for ATRP of SPE. This solvent had been used and worked very well for SI-ATRP of different monomers in our previous works.10,27,28 It had also been proven that aqueous media allow the controlled polymerization of many ionic monomers that cannot be polymerized in purely organic media.29 On the other hand, the ionic structure of the catalyst (complex of ligand and Cu(I) or Cu(II)) in aqueous solution may offer high activity for the ATRP process.30,31 Bpy was chosen as ligand in the catalyst system because this ligand has an appropriate binding constant toward Cu(I)32 and it could counteract the fast disproportionation of the Cu(I) complex in aqueous solution.33,34 As can be seen from Figure 1, with the increase of monomer concentration, the DG increased. However, without addition of CuBr2 the polymer chains propagated very fast and relatively high DG (∼2 μg/cm2) was achieved in few minutes. A quick loss of reactive chain ends could be deduced from the fact that the DG reached a plateau in one hour. This is due to the high radical concentration in the initial stage caused by lack of deactivating Cu(II)/ligand complex which leads to significant chain termination and, consequently, only a short propagating period. Cu(II) bromide

Figure 1. Dependence of polySPE DG on the surface-initiated ATRP time. (a) [SPE]/[CuBr]/[Bpy] = 10:0.5:1, (■) [SPE] = 0.3 M, (●) [SPE] = 0.1 M; (b) [SPE]/[CuBr]/[CuBr2]/[Bpy] = 50:0.5:0.05:1, (■) [SPE] = 0.3 M, (●) [SPE] = 0.1 M.

was then added to the reaction system to provide a high enough concentration of deactivating Cu(II) complex already at the beginning of the ATRP.35 After adding 10% (molar ratio, relative to CuBr) CuBr2, the DG showed a linear increase within 4 h even with low monomer concentration. Moreover, the grafted polymer chains still seemed to retain their “living” character even after 24 h. The slowdown of the overall polymerization rate can be ascribed to the burying of the reactive chain ends in the grafted polymer layer. Similar to previous work,26 no significant morphology changes were observed in scanning electron microscopy for membranes with degrees of grafting in the range obtained in this study. With permporometry it had been found that the pore diameter in dry state was reduced from 690 nm for the unmodified to about 630 nm for membranes with a degree of grafting of around 3 μg/cm2 obtained after 4 h SI-ATRP. 3.2. Zeta Potential. Zeta potentials were determined as function of pH in order to evaluate the surface properties of unmodified and polySPE functionalized membranes (Figure 2). It can be seen that the surface charge of the unmodified PET membrane was mainly negative in the pH range of 3−10 and an isoelectric point at about pH 4 was found. This isoelectric point fits well with the pKa value of benzoic acid (4.21) which is one of the two end groups in PET.36 At pH higher than 4, the zeta 2946



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Figure 2. Zeta potential vs pH for unmodified (■) and polySPE grafted (○, DG = 1.5 μg/cm2; × , DG = 3.5 μg/cm2) PET membranes.

Figure 3. Ionic strength-dependent flux of phosphate buffer solution (10 mM, pH 7.4) through unmodified (■) and polySPE-grafted PET membranes with different DG: (●) 1.0 μg/cm2, (▲) 2.6 μg/cm2, (▼) 3.9 μg/cm2.

potential became more and more negative and reached a plateau of about −28 mV at pH 7.5. This trend can be ascribed to deprotonation of carboxylic acid groups at relatively low surface density. As discussed before,24 at both high and low pH (below isoelectric point), adsorption of excess ions can also contribute to the zeta potential. After grafting of polySPE, the membrane showed much lower absolute value of zeta potential in the whole measured pH range. PolySPE is a strong−strong type zwitterionic polymer and can retain its zwitterionic property throughout the entire pH range. Therefore, surfaces fully covered with polySPE have zero net charges and absolute value of zeta potential. The membrane with lower DG still showed a considerably negative zeta potential at higher pH which can be ascribed to the incomplete coverage of the PET surface by grafted polySPE. With higher DG (3.5 μg/cm2) of polySPE grafted on the membrane pore surface, the membrane showed almost zero absolute value of zeta potential, indicating the full surface coverage with polySPE. 3.3. Effects of Salt Type and Ionic Strength on PolySPE Chain Conformation. To evaluate the influences of salt type and ionic strength on the conformation of grafted polySPE, fluxes of various salt solutions through the membrane were measured. Using the advantage of the uniform capillary pores of the PET membrane, the conformational variation of the polySPE chains grafted on the membrane pores can be sensitively monitored by detecting changes of flux. First, we measured fluxes of phosphate buffer solutions with different NaCl concentrations. As can be seen in Figure 3, the flux decreased with the increase of DG. This is due to the increase of grafted polymer layer thickness with the increase of DG. On the other hand, ionic strength (salt concentration) also significantly affected the flux (Scheme 1b). For unmodified membrane, a slight decrease in flux was observed with the increase of NaCl concentration which can be ascribed to slightly higher viscosity of NaCl solution compared with pure water. However, for polySPE grafted membranes, the flux decreased much more obviously with the increase of NaCl concentration. This result can be ascribed to the “antipolyelectrolyte” effect of grafted polySPE. Early study revealed that the antipolyelectrolyte effect of zwitterionic polymers was associated with the shielding of electrostatic interactions between ion pairs on the polymer chains.37 In pure water,

the side groups of the polySPE chains tend to adopt a cyclic conformation by association of the anionic sulfonate end group with the positively charged ammonium group. Moreover, there are also intermolecule interactions between ionic groups of neighboring polySPE chains. The presence of these strong ionic bonds forces the polySPE on the membrane pore wall to exhibit a collapsed conformation as shown in Scheme 1b. In the presence of NaCl it is believed that the Na+ and Cl− ions disrupt these electrostatic interactions by forming ion pairs and the resulting increase of net charge leads to a more stretched conformation of the polySPE chains (cf. ref 16). On the other hand, adding salt may also lead to asymmetric counterion adsorption, i.e., uneven binding of counterions to positive and negative charges on polySPE chains, and subsequently result in chain expansion.38 Therefore, the grafted polymer layer expanded with increasing salt concentration and the effective pore diameter was narrowed which resulted in a reduced flux (cf. ref 39). Moreover, the influence of salt concentration was much more pronounced for membranes with higher DG. This is because the membranes with higher DG had been obtained by longer ATRP time so that the polySPE chains were longer than those for membranes with lower DG (cf. Figure 1b). Longer chains at the same density should cause larger changes of flux. To evaluate the influence of salt type on polySPE structure, we also measured fluxes of different salt solutions for membranes with the same DG. Selected data are shown in Figure 4 (for detailed overview on flux data, please see Tables S1 and S2 in the Supporting Information) and the hydrodynamic layer thickness (HLT) of the grafted polySPE were calculated from these flux data and are shown in Figure 5. As can be seen, in pure water the polySPE layer had a thickness of ∼20 nm because of the collapsed conformation of the hydrophilic polymer chains. This thickness is very similar to the observed change of membrane pore radius in dry state (cf. Section 3.1). However, the chain structure can be varied in a specific way by adding different salts. In pure water, the flux was effectively reduced by adding chaotropic chloride and perchlorate salts. Both perchlorate and chloride salts decreased the flux gradually with the increase of salt concentration, i.e., ionic strength, in the water. However, the perchlorate salt had a 2947



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Figure 4. Flux of pure water and aqueous solutions of different salts through polySPE-grafted PET membrane with DG of 2.7 μg/cm2.

On the other hand, an opposite effect was observed with phosphate salt; the flux of 10 mM phosphate buffer was slightly higher than that of pure water (cf. Figure 4). This could be ascribed to the kosmotropic nature of the phosphate ions which have strong interactions with water and tend to “salt out” the polySPE chains. Irgum et al. reported similar effects of sodium carbonate onto zwitterionic polymer and they also attributed this to the “salting out” effect.17 Moreover, both the chloride and the perchlorate salts exhibited less effect on expanding the polySPE chains in phosphate buffer solution than in pure water (compare the solid round and square symbols with the hollow ones in Figure 5). In the presence of phosphate salts, the grafted polySPE chains showed relatively lower hydrodynamic thickness (∼50 nm) even with 1 M NaCl added to the buffer. This could be the result of opposite effects by chaotropic and kosmotropic ions in the same system. In phosphate buffer the kosmotropic phosphate ions with their strong interaction with water might compensate part of the influence of the chaotropic ions. To further confirm this effect, the flux of aqueous solution of a strong kosmotropic salt (K2SO4) was measured with other independent membrane samples. As shown in Table 1, polySPE

Figure 5. Hydrodynamic thickness of the polySPE layer grafted on PET membrane surface with DG of 2.7 μg/cm2 in pure water (▲), aqueous solutions of NaCl (■) and KClO4 (●), and phosphate buffer solution of NaCl (□) and KClO4(○); the two lowest KClO4 concentrations were 2 and 8 mM.

Table 1. Fluxes of Water with and without Small Amount of Kosmotropic Salts Through PolySPE-Grafted Membrane

very strong influence and even a very small concentration (2 mM) could significantly decrease the flux, i.e., extend the polySPE chain. The HLT of polySPE in 2 mM perchlorate salt solution was more than two times (∼43 nm) of that in pure water. Moreover, pure water with 50 mM perchlorate salt had similar flux as that of water with much higher concentration (400 mM) of chloride salt; in both cases the HLT was about 72 nm. This result indicates that the perchlorate salt has much stronger effect on polySPE conformation than chloride salt does. This follows the Hofmeister series in which ClO4− is a much stronger chaotrope than Cl−. It had been proposed that stronger chaotropes have relatively weak interactions with water molecules and interact stronger with the ammonium groups of polySPE (cf. refs 17, 40, 41). Moreover, stronger interaction between perchlorate ion and ammonium group may also lead to more obvious different counterion adsorption on positively and negatively charged groups which subsequently leads to a larger overall net charge on the polySPE chains.42 Therefore, the stronger chaotropic ion (ClO4−) was more efficient in screening electrostatic attraction on polySPE chains and resulted in a more expanded chain conformation.

flux (L/m2bar h) 2

DG (μg/cm )

water

water + 10 mM K2SO4

1.5 4.0

45370 ± 284 35467 ± 454

47068 ± 957 40148 ± 620

grafted membranes with both high and low DGs showed significantly enhanced permeability for 10 mM K2SO4 solution compared with pure water. This result indicates that the presence of SO42−‑ further “salts out” the polySPE chains from the aqueous solution, leading to a more shrunken polymer layer. 3.4. IgG Binding on PolySPE-Grafted PET Membranes. 3.4.1. Static Adsorption Mode. IgG binding was first carried out in a static way by immersing membrane samples in IgG solution for sufficient time to establish binding equilibrium and then measuring the amount of adsorbed protein. Pure water was used as solvent and various amounts of NaCl were added in order to interfere with the polySPE structure and the IgG binding. As shown in Table 2, considerable IgG binding was 2948



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Table 2. Static Adsorption of IgG on PolySPE-Grafted PET Membranes in Water with Different NaCl Concentrations

Table 3. Elution of IgG from PET Membrane in Water and Water with Different NaCl Concentrations

IgG binding (μg/cm2) in water with different NaCl concentrations (mM) DG (μg/cm2)

0

100

400

1000

3.4 5.2

0.18 ± 0.02 0.25 ± 0.04

0.08 ± 0.02 0.06 ± 0.03

0.76 ± 0.06 1.42 ± 0.09

0a 0a

a

elution in water (%) with different NaCl concentrations (mM)a DG (μg/ cm2)

IgG binding (μg/cm2) in water with 400 mM NaCl

0

100

1000

3.4 5.2

0.76 ± 0.06 1.42 ± 0.09

68.4 85

60.5 87.3

89.5 95.1

No binding detected. a

observed in pure water and this binding was significantly suppressed by adding 100 mM NaCl. When the NaCl concentration was increased to 400 mM, the membrane showed obvious IgG binding and a binding capacity of about 5 times relative to an IgG monolayer on the specific surface area was achieved. However, further increasing the NaCl concentration to 1 M led to a total loss of binding capability. In pure water the polySPE chains adopt a collapsed structure and form a relatively compact hydrated layer (cf. Figure 5). Therefore, the polymer layer acted as a surface exposing closely spaced negative and positive charges and protein could only adsorb to the surface in a two-dimensional fashion. This was manifested by the observed monolayer binding capacity. Small amounts of NaCl may “salt in” the polySPE which results in well hydrated polySPE chains. This led to a decreased binding capacity, what can also be linked to the well-known low-protein binding (“anti-fouling”) properties of such zwitterionic polymer layers (cf. ref 43). Further increasing the NaCl concentration may disturb the water structure because of the chaotropic nature of the Cl− ions and break the water clusters surrounding polySPE chains and the protein molecules: The polySPE and IgG were then partially “salted out” and bound together. Under the experimental conditions (pH 6), the IgG (isoelectric point around 7) is slightly positively charged and it is likely to bind to the negative sulfonate groups on polySPE. However, too high salt concentration (e.g., 1 M) then screened the electrostatic interactions between zwitterionic polymer and the protein and no binding was detected under this condition. Protein binding in a three-dimensional functional layer at 400 mM NaCl correlates very well with the maximum layer thickness reached at this salt concentration (cf. Figure 5). Moreover, the binding capacity of this polySPE grafted membrane is proportional with the DG (i.e., chain length). That both increases were proportional is a strong indication that the protein binding within the grafted layer is not hindered by too high grafting density (what would be expected in the “brush regime”); this is in line with the low initiator density (max. 0.2 nm−2) used in this study (cf. Section 3.1). By adjusting the chain length with ATRP time (cf. Figure 1), the binding capacity of the membrane can be tuned (cf. Table 2). To further confirm the effect of NaCl concentration on the IgG binding, we performed elution experiments. Samples with IgG bound from 400 mM NaCl aqueous solution were eluted either with pure water, water with 100 mM NaCl or water with 1 M NaCl. As can be seen from Table 3, all three solutions showed significant desorption effects, and recovery of 85−95% of IgG from the membranes with the higher DG could be achieved. These results demonstrate the possibility of binding proteins at medium salt concentration and eluting at either low (zero) or very high salt concentrations, which provide the most promising alternatives for protein purification.

Percentage of eluted protein compared with bound IgG.

3.4.2. Dynamic Flow-through Mode. To facilitate mass transfer and to maximize the binding capacity, we also performed IgG binding in flow-through mode. This is closer to the application conditions for convective macroporous membrane adsorbers. First, the conditions used in static adsorption were repeated and similar results were obtained. Slight but not negligible adsorption was observed in pure water and with the increase of NaCl concentration, the binding capacity decreased (Figure 6a). The IgG binding capacity then increased in the NaCl concentration range of 100−500 mM and reached a maximum at about 500 mM. Further increase of NaCl concentration then led to a decreased binding capacity again. Considering the DG of the samples, the binding capacity

Figure 6. IgG binding on polySPE grafted membrane with DG of 2.7 μg/cm2 in dynamic flow-through mode in presence of (a) NaCl and (b) KClO4. 2949



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complicated as we found two maximum points of binding capacity at two perchlorate salt concentrations (2 and 10 mM, cf. Figure 6b). We assume that our results could be ascribed to the influence of chaotropic ions on the solution properties of polyelectrolytes including both the polySPE and the protein. Generally speaking, for weak chaotropic salt like NaCl, relatively small amount of Cl− ions could “salt-in” the polySPE and protein which facilitates the formation of a hydration layer around these molecules. Therefore no binding could occur at this condition. Upon increasing the ion concentration to some extent, both the protein and the polySPE chains are “salted-out” from the water, thus facilitating protein binding to the grafted polySPE. This “salting out” effect will dominate in a certain salt concentration range. After that, with further increase of salt concentration a transition to “salting in” conditions will take place and the bound protein will be eluted from the polySPE chains. For strong chaotropic ion like ClO4−, very small concentration (2 mM) of this ion can effectively “salt-out” the protein and polySPE resulting in obvious binding. Further increase of the concentration of perchlorate salt leads to “salting in” effect and less binding was observed between 2 mM and 10 mM concentration range. However, the perchlorate ion, unlike the chloride ion, has a strong interaction with quaternary ammonium groups and they could form a tight ion pair which leaves the polymer layer, i.e., the “Donnan membrane”, somewhat negatively charged (cf. ref 41 and discussion above). Therefore, we still found considerable binding of positively charged IgG at 4 and 8 mM perchlorate salt concentration. When we increased the concentration to 10 mM, there were enough perchlorate ions to associate with quaternary ammonium groups and the polymer layer turned to be more negatively charged resulting in enhanced binding of IgG in a clearly three-dimensional manner. Further increase in perchlorate salt concentration then caused the “salting in” effect to become dominating again and the protein binding tended to be suppressed.

was significantly enhanced in the dynamic mode compared with that during static adsorption (cf. Table 2). Obviously, the convective flow helps the protein to penetrate the polymer layer and reach the binding sites. This is similar to observations in a recent study with macroporous membrane adsorbers comprising grafted three-dimensional glycopolymer binding layers.10 In order to reveal the influence of salt type in the solution on protein adsorption, IgG binding experiments were also carried out in water in the presence of KClO4. As can be seen from Figure 6b, KClO4 also showed evident effects on IgG binding. However, they differ from the NaCl case in that KClO4 functions in much lower and much more narrow concentration range (0−10 mM, maximum solubility in water: 129 mM), i.e., very low concentration of KClO4 can effectively influence IgG binding. This fits well with the effects of these two chaotropic ions on polySPE structure (cf. Section 3.3) and follows the Hofmeister series. Similar results were also observed by Viklund et al.16,17 They found that small concentrations of perchlorate ions strengthened the interaction between proteins and the crosslinked polySPE monolith and the increase in retention time was pronounced even with perchlorate salt concentration in the millimolar range. Moreover, further increasing perchlorate concentration to 70 mM led to the elution of the bound protein. However, their results indicated that in 5 mM phosphate buffer solution the protein uptake was continuously increased with perchlorate salt concentration from 0 to 15 mM. On the other hand, they also proved that there is no contribution from hydrophobic interaction to this protein binding property of the ZIC-HILIC phase in the presence of perchlorate salt. Furthermore, they ascribed the special protein binding property only to the influence of perchlorate salt on the zwitterionic polymer structure rather than on the protein conformation. Cook et al. had established a “chaotropic selectivity” mechanism for the ZIC phase chromatography.41 It is believed that the monovalent charge on perchlorate is delocalized over all four oxygen atoms which facilitates the interactions with basic compounds, causing strong interactions with quaternary ammonium groups in the zwitterionic species.44 Therefore, in the presence of perchlorate ion, the zwitterionic polymers exhibit a relatively strong negatively charged layer created by the terminal sulfonate groups of the zwitterion, which acts as a negative “Donnan membrane” and exerts a strong repulsive interaction with negatively charged analytes. This could be applied to explain how the perchlorate ion affects IgG binding of our case in which the IgG was positively charged in the aqueous solution (pH ≈ 6). However, in this theory, the chloride ion has much weaker interaction with the quaternary ammonium groups, causing no shielding of the positive charges on the zwitterionic polymers. Similar chaotropic effect was also reported by Sereda et al.45 They assumed that the strengthening of the interaction by perchlorate ion was the result of hydrophobicity increase at the interface of the analyte and the stationary phase. Nevertheless, this theory has a critical weakness because the experimental results showed that the increase of interactions between the target molecules and the stationary phase only occurs in certain range of concentration, and further increase of chaotropic ion concentration will result in a decrease of the binding strength. The mechanism of ZIC phase chromatography is still not well clarified and continues to be a subject of some debate.46 In our case, the situation is even more

4. CONCLUSIONS Zwitterionic polymer, polySPE, was grafted from track-etched PET membrane surface and the grafting processes via surfaceinitiated ATRP were well controlled. Using the advantage of capillary pores of the track-etched PET membrane, the conformational variation of the polySPE chains grafted on the membrane pores could be sensitively monitored by changes of flux, and the influences of salt type and ionic strength on the conformation of grafted polySPE were estimated by measuring the fluxes of various salts solutions through the membrane. All results revealed that the grafted polySPE exhibited an antipolyelectrolyte effect. Flux through the membrane was reduced by adding chaotropic chloride and perchlorate salts to the solution which extended the polySPE chains grafted on membrane pore wall. Perchlorate salt exhibited much stronger effect on polySPE chain conformation than chloride salt. On the contrary, small amounts of kosmotropic ions (10 mM SO42−) could further “salt-out” the polySPE chains and shrink the polymer layer. Hence, one very important finding was that these membranes showed pronounced stimuli-responsive behavior which was also depending on specific salt ions. The membranes were then used as membrane adsorber for IgG binding under both static adsorption and dynamic flow-through conditions. Static adsorption and dynamic experiments showed that IgG could be loaded to the membrane at medium salt concentration and bound protein could be efficiently eluted at 2950



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(14) Rana, D.; Matsuura, T. Chem. Rev. 2010, 110, 2448−2471. (15) Yang, Q.; Adrus, N.; Tomicki, F.; Ulbricht, M. J. Mater. Chem. 2011, 21, 2783−2811. (16) Viklund, C.; Irgum, K. Macromolecules 2000, 33, 2539−2544. (17) Viklund, C.; Sjogren, A.; Irgum, K.; Nes, I. Anal. Chem. 2001, 73, 444−452. (18) Boersema, P. J.; Divecha, N.; Heck, A. J.; Mohammed, S. J. Proteome Res. 2007, 6, 937−946. (19) Intoh, A.; Kurisaki, A.; Fukuda, H.; Asashima, M. Biomed. Chromatogr. 2009, 23, 607−614. (20) Jiang, W.; Fischer, G.; Girmay, Y.; Irgum, K. J. Chromatogr. A 2006, 1127, 82−91. (21) Wikberg, E.; Verhage, J. J.; Viklund, C.; Irgum, K. J. Sep. Sci. 2009, 32, 2008−2016. (22) Hemström, P.; Szumski, M.; Irgum, K. Anal. Chem. 2006, 78, 7098−7103. (23) Di Palma, S.; Boersema, P. J.; Heck, A. J. R.; Mohammed, S. Anal. Chem. 2011, 83, 3440−3447. (24) Geismann, C.; Yaroshchuk, A.; Ulbricht, M. Langmuir 2007, 23, 76−83. (25) Friebe, A.; Ulbricht, M. Langmuir 2007, 23, 10316−10322. (26) Friebe, A.; Ulbricht, M. Macromolecules 2009, 42, 1838−1848. (27) Yang, Q.; Kaul, C.; Ulbricht, M. Langmuir 2010, 26, 5746− 5752. (28) Himstedt, H. H.; Yang, Q.; Prasad Dasi, L.; Qian, X.; S. Wickramasinghe, R.; Ulbricht, M. Langmuir 2011, 27, 5574−5581. (29) Tsarevsky, N. V.; Pintauer, T.; Matyjaszewski, K. Macromolecules 2004, 37, 9768−9778. (30) Kickelbick, G.; Reinohl, U.; Ertel, T. S.; Weber, A.; Bertagnolli, H.; Matyjaszewski, K. Inorg. Chem. 2001, 40, 6−8. (31) Matyjaszewski, K.; Xia, J. H. Chem. Rev. 2001, 101, 2921−2990. (32) Sillén, L. G.; Martell, A. E. Stability Constants of Metal-Ion Complexes, Supplement No. 1; Special Publication No 25; The Chemical Society: London, 1971. (33) Tsarevsky, N. V.; Braunecker, W. A.; Matyjaszewski, K. J. Organomet. Chem. 2007, 692, 3212−3222. (34) Tsarevsky, N. V.; Matyjaszewski, K. Chem. Rev. 2007, 107, 2270−2299. (35) Matyjaszewski, K.; Miller, P. J.; Shukla, N.; Immaraporn, B.; Gelman, A.; Luokala, B. B.; Siclovan, T. M.; Kickelbick, G.; Vallant, T.; Hoffmann, H.; Pakula, T. Macromolecules 1999, 32, 8716−8724. (36) Hsieh, Y.-Y.; Lin, Y.-H.; Yang, J.-S.; Wei, G.-T.; Tien, P.; Chau, L.-K. J. Chromatogr. A 2002, 952, 255−266. (37) Niu, A.; Liaw, D.-J.; Sang, H.-C.; Wu, C. Macromolecules 2000, 33, 3492−3494. (38) Kumar, R.; Fredrickson, G. H. J. Chem. Phys. 2009, 131, 104901. (39) Zhao, Y.; Wee, K.; Bai, R. ACS Appl. Mater. Interfaces 2010, 2, 203−211. (40) Wikberg, E. Zwitterionic Sulfobetaine Polymers as Stationary Phases for Liquid Chromatography, Ph.D. Dissertation, Umeå University, Sweden, 2008. (41) Cook, H. A.; Hu, W.; Fritz, J. S.; Haddad, P. R. Anal. Chem. 2001, 73, 3022−3027. (42) Dobrynin, A. V.; Rubinstein, M. J. Phys. II 1995, 5, 677−695. (43) Susanto, H.; Ulbricht, M. Langmuir 2007, 23, 7818−7830. (44) Haddad, P. R.; Jackson, P. E. Ion Chromatography: Principles and Applications; Elsevier: Amsterdam, 1990. (45) Sereda, T. J.; Mant, C. T.; Hodges, R. S. J. Chromatogr. A 1997, 776, 153−165. (46) Fritz, J. S. J. Chromatogr. A 2005, 1085, 8−17.

either low (zero) or very high salt concentrations. Both chaotropic KClO4 and NaCl showed similar effects on IgG binding: Suppression of binding at very low concentration, then increasing binding to maximum capacity, followed by binding suppression at very high concentrations. However, KClO4 functions in a much lower and much narrower concentration range, which follows the Hofmeister series. Especially the possibility of protein elution at low (zero) salt concentration is very promising for protein purification: (Gradient) elution at very low buffer salt concentration will reduce the effort for buffer exchange or removal in subsequent steps. Tuning the maximum binding capacity by specific salts has significant potential because the protein binding selectivity can be increased. The binding capacity of the membrane could be controlled by adjusting the chain length of grafted polySPE. Protein binding in three-dimensional grafted functional layers is attractive because porous membranes with high static capacity can be achieved; a high dynamic capacity is ensured by convective flow through pores with a radius, which is much larger than hydrodynamic layer thickness. Overall, the results of this study with a model pore membrane have significant implications for the development of novel stimuli-responsive pore systems and materials and of efficient macroporous membrane adsorbers for protein purification.

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ASSOCIATED CONTENT

S Supporting Information *

Pore size distribution of the base membrane and detailed flux data obtained under various salt conditions can be found in Supporting Information. This information is available free of charge via the Internet at http://pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support for the work by the Bundesministerium für Forschung und Technologie, Germany (BMBF; grant no. 315339B) is gratefully acknowledged.

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REFERENCES

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Novel Membrane Adsorbers with Grafted Zwitterionic Polymers

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