Intermolecular Forces between Proteins and Polymer Films with

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Intermolecular Forces between Proteins and Polymer Films with Relevance to Filtration Jeffrey A. Koehler, Mathias Ulbricht,† and Georges Belfort* Rensselaer Polytechnic Institute, Howard P. Isermann Department of Chemical Engineering, Troy, New York 12180-3590 Received January 3, 1997X

In order to understand the effects of protein fouling during ultrafiltration of biological fluids, we have investigated the molecular interactions between a thin polysulfone film and hen egg lysozyme with the surface forces apparatus (SFA). The normalized forces between the adsorbed protein layers and polymer films were measured below, at and above the pI of lysozyme, and compared with four different permeation fluxes obtained from ultrafiltration experiments. The intermolecular forces between two protein layers were also measured at the different pH values. Adsorption kinetics of lysozyme onto mica were also obtained. Buffer and lysozyme solutions at similar pH values and concentrations were filtered with 6 kD polysulfone membranes to obtain flux decline and hence fouling measurements. Hydrophobic membranes, such as polysulfone, exhibit extremely long-range attractive interactions (on the order of 1500-2000 Å) with proteins such as lysozyme. Even in the presence of electrostatic repulsion at pH values above the isoelectric point of lysozyme (when both lysozyme and polysulfone were negatively charged), a long-range attractive interaction of around 210 µN/m was observed. Such interactions were absent with measurements between adsorbed lysozyme-lysozyme layers. From these measurements, simple linear correlations were found relating the normalized forces to the fluxes from the ultrafiltration experiments. With respect to fouling, protein-protein and protein-polymer interactions are about equally important during ultrafiltration. This suggests that both the surface chemistry of the membrane and the solution conditions could be chosen to minimize fouling for specific protein solutions. Hence, as a result of this study, fouling of polysulfone membranes with lysozyme solutions can be reduced if (i) filtration is conducted at pH values above the pI of lysozyme (approximately 10.8) and (ii) the membranes are modified such that the longrange attractive interactions are reduced. These results support those from previous phenomenological studies on membrane filtration of protein solutions and are the first evidence relating intermolecular force interactions with macroscopic events in membrane fouling.

Introduction Membrane fouling is composed of pore plugging, pore narrowing, and cake deposition. All of these phenomena share two important factors in the filtration of biological fluids: the positive interactions between dissolved protein and itself (aggregation) and between dissolved protein and the membrane surface (adsorption). The mechanisms that underlie these attractive forces at the molecular level are unknown. This is significant because, in principle, membranes could be produced that exhibit smaller attractive forces between the membrane surface and the protein. This, in turn, should yield membranes that have a longer operational life and exhibit higher performance characteristics (i.e. retention and flux). To date, a great deal of work has been performed on membrane filtration using various macromolecules including proteins (such as bovine serum albumin, BSA), dextrans, polyethylene glycol, and others. Different fluxdecline rates occurred for all the various cases, showing a solute dependent process. Thus, each new membrane or macromolecule system must be individually analyzed. Among the many reasons that have been proposed for this result are the following: (1) protein adsorption (specifically protein-membrane and protein-protein interactions and their dependence on pH and ionic strength,1-6 (2) reduced driving force due to an osmotic back-pressure from solute buildup at the membrane † Current Address: Fachbereich Chemie, Institut fu ¨ r Organische und Bioorganische Chemie, Humboldt-Universita¨t zu Berlin, Berlin, Germany. * Corresponding author. Telephone: (518) 276-6948. Fax: (518) 276-4030. E-mail: [email protected]. X Abstract published in Advance ACS Abstracts, July 1, 1997.

S0743-7463(97)00010-3 CCC: $14.00

surface,7-12 (3) increased resistance due to protein deposition and cake formation along with the increased viscosity of the fluid near the membrane surface.13-17 (1) Fane, A. G.; Fell, C. J. D.; Suki, A. The effect of pH and ionic environment on the ultrafiltration of protein solutions with retentive membranes. J. Membr. Sci. 1983, 16, 195. (2) Mattiasson, E. The role of macromolecular adsorption in fouling of ultrafiltration membranes. J. Membr. Sci. 1983, 16, 23. (3) Nystrom, M.; Laatikainen, M.; Turku, M.; Jarvinen, P. Resistance to fouling accomplished by modification of ultrafiltration membranes. Prog. Colloid Polym. Sci. 1990, 82, 321. (4) McDonogh, R.; Bauser, H.; Stroh, N.; Chmiel, H. Concentration polarization and adsorption effects in cross-flow ultrafiltration of proteins. Desalination 1990, 79, 217. (5) Robertson, B. C.; Zydney, A. L. Protein adsorption in asymmetric ultrafiltration membranes with highly constricted pores. J. Colloid Interface Sci. 1990, 134 (2), 563. (6) Meireles, M.; Aimer, P.; Sanchez. Albumin denaturation during ultrafiltration: effects of operating conditions and consequences on membrane fouling. V. Biotechnol. Bioeng. 1991, 38, 528. (7) Nabetani, H.; Nakajima, M.; Watanabe, A. Effects of osmotic pressure and adsorption on ultrafiltration of ovalbumin. AICHE J. 1990, 36 (6), 907. (8) Goldsmith, R. L. Macromolecular ultrafiltration with microporous membranes. Ind. Eng. Chem. Fundam. 1971, 10, 113. (9) Leung, W. F.; Probstein, R. F. Low polarization in laminar ultrafiltration of macromolecular solutions. Ind. Eng. Chem. Fundam. 1979, 18 (3), 274. (10) Vilker, V. L.; Colton, C. K.; Smith, K. The osmotic pressure of concentrated protein solutions. Effect of concentration and ph in saline solutions of bovine serum albumin. J. Colloid Interface Sci. 1981, 79 (2), 548. (11) Jonsson, G. Boundary Layer Phenomena during the Ultrafiltration of Dextran and Whey Protein Solutions. Desalination 1984, 51, 61. (12) Wijmans, J. G.; Nakao, S.; Smolders, C. A. Flux limitation in ultrafiltration: osmotic pressure and gel layer model. J. Membr. Sci. 1984, 20, 115. (13) Blatt, W. F.; Dravid, A.; Michaels, A. S.; Nelson, L. Solute polarization and cake formation in membrane ultrafiltration. Causes, consequences and control techniques. In Membrane Science and Technology; Flinn, J. E., Ed.; Plenum Press: New York, New York, 1970; pp 47-97.

© 1997 American Chemical Society

Forces between Proteins and Polymer Films

With BSA as a model protein, experiments have shown that fluxes are higher when the pH of the solution is not equal to the pI of the protein,1,3,4,14,17 when the ionic strength of the solution is low,14 and when the surface of the membrane is hydrophilic.18 In several recent papers, Zydney and co-workers19,20 and Tracey and Davis21 have proposed a two-step mechanism to describe BSA fouling and flux decline in stirred cell microfiltration. Large BSA aggregates are convectively dragged toward the membrane surface constricting and blocking the pores, with these aggregates serving as nucleation or attachment sites for further BSA deposition. By filtering (i) prefiltered BSA solutions or (ii) nonaggregating BSA (cysteinyl- and carboxymethyl-mediated cap of the free sulfhydryl group on BSA), Kelly and Zydney20 have shown that minimal flux decline occurs. When they used a prefiltered BSA solution, they were able to show that, in the absence of aggregates, there was little, if any, fouling. Also, the rate of mixing and hence back-migration had no effect on the results. With mixtures of unfiltered and prefiltered BSA (through a 100 kDa molecular weight cutoff ultrafiltration membrane), they showed that “flux-decline was determined entirely by the deposition of aggregates and was unaffected by the concentration of native (monomeric) BSA”.20 Kim and Fane22 have compared the performance of four commercial ultrafiltration (UF) membranes with the same nominal molecular weight cutoff but with different hydrophilicities using a 0.1 wt % BSA solution in a crossflow test cell. They obtained “enhanced UF fluxes with slower flux loss and lower solute resistance” for the hydrophilic membranes as compared to the unmodified hydrophobic membranes (as measured by contact angle). They also report that the “hydrophilic membranes were not necessarily easier to clean”. In addition, they observed, as others have before, that UF fluxes were always greater when the solution pH was away from the pI. Maa and Hsu23 provide convincing evidence that the presence of aggregates in solution is not the only potential cause of fouling with recombinant human growth hormone (rhGH). They suggest that “aggregation/adsorption in the filter pores during filtration is a better explanation for membrane fouling”. High pH, low salt, and the presence of a nonionic detergent all resulted in improved flux. These phenomenological studies are extremely useful in suggesting the causes of fouling, since they provide indirect evidence of the effects of fouling. To understand (14) Fane, A. G.; Fell, C. J. D.; Waters, A. G. Ultrafiltration of protein solutions through partially permeable membranes. The effect of adsorption and solution environment. J. Membr. Sci. 1983, 16, 211. (15) Nakao, S.-I.; Nomura, T.; Kimura, S. Characteristics of macromolecular gel layer formed on ultrafiltration tubular membrane. AICHE J. 1979, 25, 615. (16) Nakao, S.-I.; Kimura, S. Analysis of solute rejection in ultrafiltration. J. Chem. Eng. Jpn. 1981, 14 (1), 32. (17) Tirmizi, N. P. Study of ultrafiltration of macromolecular solutions. PhD Thesis, Department of Chemical Engineering, Rennselaer Polytechnic Institute, Troy, NY, 1990. (18) Hannemaaijer, J. H.; Robbertsen, T.; van den Boomgaard, Th.; Gunnink, J. W. Characterization of clean and fouled ultrafiltration membranes. Desalination 1988, 68, 93. (19) Kelly, S. T.; Opong, W. S.; Zydney, A. L. The influence of protein aggregates on the fouling of microfiltration membranes during stirred cell filtration. J. Membr. Sci. 1995, 80, 175. (20) Kelly, S. T.; Zydney, A. L. Mechanisms for BSA fouling during microfiltration. J. Membr. Sci. 1995, 107, 115. (21) Tracey, E. M.; Davis, R. H. BSA fouling of track-etched polycarbonate microfiltration membranes. J. Colloid Intferface Sci. 1994, 167, 104. (22) Kim, K.-J.; Fane, A. G. Performance evaluation of surface hydrophilized novel ultrafiltration membranes using aqueous proteins. J. Membr. Sci. 1995, 99, 149. (23) Maa, Y.-F.; Hsu, C. C. Membrane fouling in sterile filtration of recombinant human growth hormone. Biotechnol. Bioeng. 1996, 50, 319.

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the mechanisms and to provide direct evidence of fouling, intermolecular force measurements were performed between a model protein (lysozyme) and a thin, hydrophobic polymer film (polysulfone). Polysulfone was chosen as the polymer because it is a commonly used membrane material in industry due to its chemical and structural stability. The forces were compared with four permeation flux criteria from membrane filtration results in an attempt to relate the molecular scale measurements with the macroscopic observations. A correlation is presented that shows a simple relation between the forces and flux decline. Materials and Methods Protein. Lysozyme, Lz (Pharmacia Biotech (Lot #4100267011, Piscataway, NJ), was chosen for this experiment because it is not readily denatured and has an intermediate adiabatic compressibility value. The adiabatic compressibility, βs, of lysozyme is 4.67 × 10-12 cm2/dyn.24 This is a median value between very rigid proteins such as cytochrome c (βs ) 0.066 × 10-12 cm2/dyn) and very flexible proteins such as BSA (βs ) 10.5 × 10-12 cm2/dyn).24 Lysozyme has a prolate spheroid shape with the dimensions 30 × 30 × 45 Å3. It has a molecular mass of 14 400 Da with 129 amino acids and 4 disulfide bonds, and the isoelectric point is 10.8-11.4 with a mean about 11.1. From the space-filled model of the Lz crystal structure (not shown), it can readily be seen that Lz has both hydrophobic and hydrophilic amino acids exposed to the exterior.25 Film and Membrane. Both the films (Udel 3500, Union Carbide, Danbury, CT) and the membranes were made of polysulfone, the chemical structure of which is given by

The polysulfone ultrafiltration membranes were of the type gr81pp from Dow Danske (Nakskov, Denmark) and have a nominal molecular weight cutoff (MWCO) of 6 kDa. The films were prepared by spin-coating a drop of a 3% (w/v) solution of polysulfone dissolved in 1,2-dichlorobenzene onto mica which had been glued onto the SFA lens (see below). It was spun at a rate of 500 rpm for 5 s on a spin-coating apparatus (Photoresist spinner, Headway Co., Garland, TX) followed immediately at a rate of 5000 rpm for 40 s to smooth out the film. Drying was done in a convection oven for 2 h at 80 °C. Solvent selection was crucial to achieve both continuous homogeneous films and films strongly adhering to the mica surface. Films formed under these spin-coating conditions had a reproducible thickness of 430 ( 15 Å, measured via ellipsometry. For the ellipsometry measurements (Model II-004, Rudolph, Fairfield, NJ), the polysulfone was spun onto a silicon wafer for easy determination of the film thickness. Surface Characterization. The films and membranes were studied by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) and atomic force microscopy (AFM) to determine the chemical nature and morphology of the surfaces. ATR-FTIR was used to confirm that both surfaces were chemically similar. AFM was used to ascertain if the film surface was smooth enough to be used in the SFA; i.e., the surface roughness should not be much greater than about 10 Å. ATRFTIR spectra were taken using a 45° germanium crystal on a Nicolet Magna-IR 550 Spectrometer Series II with Auxiliary Experiment Module (Madison, WI). The AFM scans were performed in noncontact mode using a 0.2 µm ultralever on an Autoprobe CP (Park Scientific Instruments, Sunnyvale, CA). The probe tip was Si3N4 and had a tip diameter of approximately 100 (24) Kharakoz, D. P.; Sarvazyan, A. P. Hydrational and intrinsic compressibilities of globular proteins. Biopolymer 1993, 33, 11. (25) Wilson, K. P.; Malcolm, B. A.; Matthews, B. W. Structural and thermodynamic analysis of compensating mutations within the core of chicken egg white lysozyme. J. Biol. Chem. 1992, 267, 10842.

4164 Langmuir, Vol. 13, No. 15, 1997 Å. For the captive bubble contact angle measurements, small diameter air bubbles (approximately 2-3 mm) were injected from a syringe into a glass chamber containing deionized water.26 The bubble was released from the tip of a needle and floated to the surface of the inverted immersed membranes and films. The contact angles were then measured using a SIT camera (SIT66, Dage-MTI Inc., Michigan City, IN) with a lens and observed on a video screen. The values for the contact angles were averaged over four to six different air bubbles. In order to measure advancing and receding angles, the air bubbles were inflated and deflated with air and the respective contact angles were measured. The glass chamber was washed with DI water while the syringe and the needle were washed with acetone and then rinsed carefully with water before use. Surface Forces Apparatus (SFA). The SFA was used to measure intermolecular forces between the two layers; one, an adsorbed layer of lysozyme, and the other, a thin film of polysulfone or another adsorbed layer of lysozyme. The layers were adsorbed onto mica that had been glued to half-cylindrical silica lenses. The distance between the two layers was determined by interferometry. This separation was used with a known spring force constant to give the forces. The method has been described previously.27 It has been used to measure adhesion and forces between inorganic surfaces,28,29 proteins,30-39 surfactants,40-43 polymers,33,44-46 glycolipids,47 biological ligands,48 and thin hydrophobic surfaces.24,49-53 In our experiment, the surfaces were submerged in a 10-2 M KOH/HNO3 solution in a (26) Hamilton, W. C. A technique for the characterization of hydrophilic surfaces, J. Colloid Interface Sci. 1972, 40, 219-222. (27) Israelachvili, J. N.; Adams, G. E. Measurement of forces between two mica surfaces in aqueous electrolyte solutions in the range 1-100 nm. J. Chem. Soc., Faraday Trans. 1978, 74, 975. (28) Ke´kicheff, P.; Ninham, B. W. The double layer interaction in asymmetric electrolytes. Europhys. Lett. 1990, 12, 471. (29) Ducker, W. A.; Xu, Z.; Clarke, D. R.; Israelachvili, J. N. Forces between alumina surfaces in salt solutions: non-DLVO forces and the implications for colloidal processing. J. Am. Ceram. Soc. 1994, 77, 437. (30) Afshar-Rad, T.; Bailey, A. I.; Luckham, P. F.; MacNaughtan, W.; Chapman, D. Forces between proteins and model polypeptides adsorbed on mica surfaces. Biochim. Biophys. Acta 1987, 915, 101. (31) Lee, C. S.; Belfort, G. Changing activity of ribonuclease a during adsorption: a molecular explanation. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 8392. (32) Belfort, G.; Lee, C. S. Attractive and repulsive interactions between and within adsorbed ribonuclease a layers. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 9146. (33) Luckham, P. F.; Ansarifar, M. A. Biomedical aspects of the direct measurement of the forces between adsorbed polymers and proteins. Br. Polym. J. 1990, 22, 233. (34) Gallinet, J.-P.; Gauthier-Manuel, B. Structural transitions of concanavalin A adsorbed onto bare mica plates: surface force measurements. Eur. Biophys. J. 1993, 22, 195. (35) Blomberg, E.; Claesson, P. M.; Tilton, R. D. Short-range interaction between adsorbed layers of human serum albumin. J. Colloid Interface Sci. 1994, 166, 427. (36) Leckband, D. E.; Schmitt, F.-J.; Israelachvili, J. N.; Knoll, W. Direct force measurements of specific and nonspecific protein interactions. Biochemistry 1994, 33, 4611. (37) Nylander, T.; Ke´kicheff, P.; Ninham, B. W. The effect of solution behavior of insulin on interactions between adsorbed layers of insulin. J. Colloid Interface Sci. 1994, 164, 136. (38) Pincet, F.; Perez, E.; Belfort, G. Do denatured proteins behave like polymers? Macromolecules 1994, 27, 3424. (39) Pincet, F.; Perez, E.; Belfort, G. Molecular interactions between proteins and synthetic membrane polymer films. Langmuir 1995, 11, 1229. (40) Christenson, H. K.; Claesson, P. M.; Parker, J. L. Hydrophobic attraction: A reexamination of electrolyte effects. J. Phys. Chem. 1992, 96, 6725. (41) Tsao, Y.-H.; Wennerstro¨m, H.; Evans, D. F. Long-range attraction between a hydrophobic surface and a polar surface is stronger than that between two hydrophobic surfaces. Langmuir 1993, 9, 779. (42) Waltermo, A.; Sjo¨berg, M.; Anhede, B.; Claesson, P. M. Adsorption of an ethoxylated amine surfactant on mica and its effect on the surface forces. J. Colloid Interface Sci. 1993, 156, 365. (43) Mao, G.; Tsao, Y.-H.; Tirrell, M.; Hessel, V.; van Esch, J.; Ringsdorf, H.; Davis, H. T. Interactions, structure, and stability of photoreactive bolaform amphiphile multilayers. Langmuir 1995, 11, 942. (44) Argillier, J.-F.; Ramachandran, R.; Harris, W. C.; Tirrell, M. Polymer-surfactant interactions studied with the surface force apparatus. J. Colloid Interface Sci. 1991, 146, 242. (45) Kuhl, T. L.; Leckband, D. E.; Lasic, D. D.; Israelachvili, J. N. Modulation of interaction forces between bilayers exposing short-chained ethylene oxide headgroups. Biophys. J. 1994, 66, 1479.

Koehler et al. Teflon bath. The ionic strength of the solution remained the same while the pH of the solution was changed from experiment to experiment by mixing proper amounts of 10-2 M KOH or 10-2 M HNO3. The room temperature was controlled at 21 ( 1 °C. Details of the SFA used in this study have been given previously.31,32 The hard wall interaction was chosen as the zero distance reference point for all of the SFA measurements so that all of the results could be easily compared. Adsorption Kinetics. The adsorption kinetics of lysozyme onto mica were performed using ATR-FTIR. The amplitude of the amide II peak (1550 cm-1) was related to the amplitude of the mica peak at 950 cm-1 to determine the lysozyme concentration.54-56 A calibration curve was measured by placing known quantities of lysozyme solution on a specific size of mica and allowing all of the water to evaporate forcing all of the lysozyme to remain on the surface. The adsorption kinetics were measured at the four different pH values with respect to time. Filtration Measurements. The filtration experiments were performed in a thin channel crossflow membrane apparatus (TCF2, Amicon, Danvers, MA). The polysulfone membranes were received from the manufacturer already wetted in an aqueous solution containing glycerol, propionic acid, and caustic soda. The membranes were cut from the sheet and placed in deionized water in the refrigerator for 12 h to ensure complete wetting. The membrane cell was cleaned with 1 L of sodium hydroxide solution (pH 12), 1 L of hydrochloric acid solution (pH 2), 1 L of sodium hydroxide solution (pH 12), and 2 L of deionized water. After the cell was cleaned, the membrane was inserted into the cell and the buffer at which the experiment was to be run was passed through the membrane under 0.3 MPa of N2 pressure for 5 h with a 1 m/s crossflow velocity until the steady state water flux, Jw1, was achieved. The water was then removed from the cell and 10 mL of the buffer was added followed by 200 mL of the protein solution (50 mg/L) under crossflow. The cell was once again placed under 0.3 MPa of N2 pressure until 50 mL of permeate was collected. Less than 50 mL of permeate was collected for the cases where the membrane was extremely fouled. For these cases, a filtration time was used similar to that for the case where 50 mL of permeate was collected. The permeate and solute fluxes at the end of the protein filtration period are denoted by Jp and Js, respectively. The cell was drained and rinsed twice with the buffer solution and then filled again with the buffer to determine the recovery of the initial flux. The steady state flux measurement for this time period is denoted by Jw2. A typical run denoting the different flux values is given in Figure 1.

Results Characterization of Polymer Surfaces. In order to compare the force-distance measurements using polysul(46) Mangipudi, V.; Pocius, A. V.; Tirrell, M. Direct measurement of the surface energy of corona-treated polyethylene using the surface forces apparatus. Langmuir 1995, 11, 19. (47) Luckham, P.; Wood, J.; Swart, R. The surface properties of gangliosides: ii. direct measurement of the interaction between bilayers deposited on mica surfaces. J. Colloid Interface Sci. 1993, 156, 173. (48) Leckband, D. E.; Kuhl, T.; Wang, H. K.; Herron, J.; Mu¨ller, W.; Ringsdorf, H. 4-4-20 Anti-fluorescyl igg fab’ recognition of membrane bound hapten: direct evidence for the role of protein and interfacial structure. Biochem. 1995, 34, 11467. (49) Wood, J.; Sharma, R. Interaction forces between hydrophobic mica surfaces. J. Adhesion Sci. Technol. 1995, 9, 1075. (50) Wood, J.; Sharma, R. How long is the long-range hydrophobic attraction? Langmuir 1995, 11, 4797. (51) Ducker, W. A.; Xu, Z.; Israelachvili, J. N. Measurements of hydrophobic and dlvo forces in bubble-surfactant interactions in aqueous solutions. Langmuir 1994, 10, 3279. (52) Claesson, P. M.; Christenson, H. K. Very long range attractive forces between uncharged hydrocarbon and fluorocarbon surfaces in water. J. Phys. Chem. 1988, 92, 1650. (53) Herder, P. C. Forces between hydrophobed mica surfaces immersed in dodecylammonium chloride solution. J. Colloid Interface Sci. 1990, 134, 336. (54) Brash, J. L.; Lyman, D. J. Adsorption of plasma proteins in solution to uncharged, hydrophobic polymer surfaces. J. Biomed. Mater. Res. 1969, 3, 175. (55) Giroux, T. A.; Cooper, S. L. FTIR/ATR studies of human fibronectin adsorption onto plasma derivatized polystyrene. J. Colloid Interface Sci. 1990, 139, 351. (56) Fu, F.-N.; Fuller, M. P.; Singh, B. R. Use of fourier transform infrared/attenuated total reflectance spectroscopy for the study of surface adsorption of proteins. Appl. Spectrosc. 1993, 47, 98.

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Figure 1. Typical flux-time curve, showing flux designations used for filtration experiments. Jw1 is the steady state buffer flux (b) after 5 h at a 1 m/s crossflow at 0.3 MPa of pressure; Jp is the steady state protein solution flux (9) after 50 mL of 50 mg/L solution permeated the membrane at a 1 m/s crossflow at 0.3 MPa of pressure; Jw2 is the steady state buffer flux (2) after about 100 min at a 1 m/s crossflow at 0.3 MPa of pressure. All these parameters are read on the left hand ordinate. The calculated solute flux (0) estimated from the solution flux, the feed concentration, and the solute rejection is read on the right hand ordinate.

Figure 3. AFM scans of film and membrane taken in noncontact mode. The cantilever was a 0.2 µm ultralever. The probe tip was Si3N4 and had a tip diameter of approximately 100 Å. (a, top) Polysulfone film: median height 15 Å, RMS roughness 2.6 Å, average roughness 2.1 Å. (b, bottom) Polysulfone membrane: median height 67 Å, RMS roughness 9.6 Å, average roughness 7.6 Å. Table 1. Captive Air Bubble Contact Angle Measurements in Water

Figure 2. ATR-FTIR spectra of (a) the polysulfone film (Udel 3500, Union Carbide, Danbury, CT) and (b) the polysulfone membrane (gr81pp, Dow Dansk, Naskov, Denmark). Spectra were taken with a 45° Ge crystal.

fone films with the filtration results using commercial polysulfone membranes, it is necessary to show that the films and membranes are similar in chemical composition. To this end, we used the ATR-FTIR technique to probe the chemical nature of the surfaces at penetration depths of approximately 0.5-1.0 µm. Gross estimates of the surface polarity were determined by contact angle measurements. The ATR-FTIR scans for the polysulfone membrane and film are shown in Figure 2. The two scans illustrate how similar the two materials were. The aliphatic CH3 stretching is seen at 3000-2800 cm-1, and the aromatic CH stretching appears at 3100-3000 cm-1. The strong aromatic ether peak can been seen at ca. 1240 cm-1. The symmetric and asymmetric sulfone stretches can be seen at ca. 1160 and ca. 1328 cm-1, respectively. The fingerprint region (1600-800 cm-1) also shows good correlation between the two samples. Figure 3a shows the AFM scan of the polysulfone film that was spun on a flat piece of mica. The film had an average roughness of 2.1 Å and a RMS (root mean square) roughness of 2.6 Å with a mean height of 15 Å. These values were smooth enough for SFA measurements. The AFM image of the surface of the gr81pp polysulfone membrane is shown in Figure 3b.

captive bubble contact angles (air/water) material

static Θ (deg)

receding Θ (deg)

advancing Θ (deg)

cleaved mica PSU (membrane) PSU (film)

21 ( 1 99 ( 4 95 ( 1

21 ( 1 105 ( 4 106 ( 2

19 ( 1 82 ( 4 88 ( 2

The membrane had an average roughness of 7.6 Å and an RMS roughness of 9.6 Å with a mean height of 67 Å. Both surfaces were also characterized using captive bubble contact angle measurements with a bubble of air in water (Table 1). The values of the contact angles were similar, and the hystereses or differences between the advancing and receding contact angles were close to 20°. The closeness of these values is not surprising, since the AFM images have shown that both the film and membrane are fairly smooth and the ATR-FTIR images have shown that they have similar chemistry. The contact angle for mica exhibited almost no hysteresis, as expected. It is expected that the static angle values are about halfway between the values of the advancing and receding angles, and this was found to be so. These results indicate that the film and membrane surfaces are similar. Therefore, it is possible to compare the intermolecular forces between the polysulfone film and the adsorbed Lz layer with the ultrafiltration measurements of Lz solutions. In the SFA, the contact area was about 1 µm by 1 µm; these dimensions were much larger than the height fluctuations of the surface as measured by the AFM.

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Table 2. Maximum Adsorbed Amounts of Lysozyme on Freshly Cleaved Mica and Early-Time Diffusion Coefficients of Lysozyme in Solutiona pH

maximum adsorbed amount (mg/m2)

measured diffusion coefficientb (×10-7 cm2/s)

6.6 11.0 (pI) 11.6 12.0

0.83 1.00 0.79 0.46 (5)

0.848 3.28 9.08 4.56

a Lysozyme concentration: 50 mg/L. T ) 21 °C. 10-2 M KOH/ HNO3 buffer. b Creighton58 reports a value of 11.3 × 10-7 cm2/s for the diffusion coefficient. The diffusion coefficients were obtained from the slopes of the kinetic data (not shown), as described by Amiel et al.57

Lz-Mica Adsorption Kinetics and Amount. From the kinetic experiments, the amount of adsorbed Lz reached a plateau (maximum) in less than 30 min for all four pH values (6.6, 11.0, 11.6, and 12.0). The time frame of 4 h for the adsorption of the Lz in the SFA experiments was well above the time required to reach a plateau value. Similarly, all the filtration experiments with protein in the feed were longer than 2 h. Above the pI of Lz (pH 12.0), the maximum adsorbed amount was less than half (54% drop) that at the pI. This was probably due to the repulsive nature between the negatively charged mica and the net negatively charged Lz. The maximum adsorbed amount of Lz on mica was at the pI of the protein, possibly due to the hydrophobic interactions. Below the pI of Lz, adsorption was about 17% less than that at the pI but still larger than that above the pI. This was likely due to the attractive electrostatic interactions between the negatively charged mica and the positively charged Lz. The maximum adsorbed amounts (equilibrium) for all four pH values together with their initial diffusion coefficients are summarized in Table 2. The diffusion coefficients were obtained from a linear plot of adsorbed amount, A(t), versus the square root of time, according to the following equation:57

A(t) )

2 C0(Dt)1/2 π1/2

(1)

where C0 and D are the solution concentration of protein (mg/L) and the mutual diffusion coefficient (cm2/s), respectively. Since all the diffusivity values reported in Table 2 are below the single molecule diffusion coefficient of 11.3 × 10-7 cm2/s,57 we suspect aggregation to have occurred. Adsorption and desorption of Lz on silica surfaces does not change the molecule’s conformation greatly.59 The adsorption of Lz onto graphite showed an ordered twodimensional array of adsorbed lysozyme, whereas adsorption of Lz on mica was seen to exhibit two-dimensional structures at low lysozyme concentrations60 and threedimensional aggregation at higher concentrations.61,62 (57) Amiel, C.; Sikka, M.; Schneider, J. W.; Tsao, Y. H.; Tirrell, M.; Mays, J. W. Adsorption of hydrophilic-hydrophobic block copolymers on silica from aqueous solutions. Macromolecules 1995, 28, 3125. (58) Creighton, T. E. Proteins: Structures and Molecular Properties, 2nd ed.; W. H. Freeman & Co.: New York, 1993; p 266. (59) Norde, W.; Favier, J. P. Structure of adsorbed and desorbed proteins, Colloid Surf. 1992, 64, 87. (60) Haggerty, L.; Lenhoff, A. M. Analysis of ordered arrays of adsorbed lysozyme by scanning tunneling microscopy. Biophys. J. 1993, 64, 886. (61) Tilton, R. D.; Blomberg, E.; Claesson, P. M. Effect of anionic surfactant on interactions between lysozyme layers adsorbed on mica. Langmuir 1993, 9, 2102. (62) Blomberg, E.; Claesson, P. M.; Fro¨berg, J. C.; Tilton, R. D. Interaction between adsorbed layers of lysozyme studied with the surface force technique. Langmuir 1994, 10, 2325.

PSU-Lz Force Measurements. Before the PSULz forces were measured, it was necessary to determine the stability of the film in the SFA in aqueous solution and whether it remained adhered to the curved mica surface. Thus, the interaction between the PSU film and mica was measured. Since the slopes of the compression and decompression curves followed a straight line on a semilog plot and were similar and hysteresis between the curves was absent for all four pH values (two above the pI, one at the pI, and one below the pI; data not shown), the film was considered stable and well formed. The repulsion curves were linear for long distances, suggesting electrostatic repulsion between the two layers. Since the DLVO theory is not applicable for dissimilar surfaces and since we did not know the potential or charge at each of the surfaces, nor did we know if the system was operating at constant charge or potential, a nonlinear PoissonBoltzmann theory for calculating the electric double-layer force and interaction free energy between dissimilar charged surfaces was not used.63 Next, Lz was adsorbed onto the mica of one surface at the respective pH values and the PSU-Lz interactions were measured (Figure 4). In all cases, a small attractive interaction (