Immobilization and Characterization of Poly(acrylic acid) - American

Langmuir 2002, 18, 10137-10145

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Immobilization and Characterization of Poly(acrylic acid) Graft Layers Patrick Vermette†,‡,§ and Laurence Meagher*,†,‡ CSIRO Molecular Science, Bag 10, Clayton South, VIC 3169, Australia, and Cooperative Research Centre for Eye Research and Technology (CRCERT), Rupert Myers Building, The University of New South Wales, Sydney, NSW 2052, Australia Received March 22, 2002. In Final Form: September 30, 2002 This study reports the covalent immobilization of poly(acrylic acid) (PAAC) onto thin films deposited using radio-frequency glow discharge from a vapor of n-heptylamine (HApp). The successful immobilization of PAAC onto HApp was demonstrated by XPS analyses and AFM colloid probe force measurements. The force profiles obtained between silica particles and the PAAC coating were repulsive in nature, roughly exponentially decaying and of long range. The interaction measurements for the PAAC surfaces were not purely electrostatic in nature but also a result of compression of the covalently attached PAAC layer by the silica surface (i.e., electrosteric). The concentration of EDC/NHS added during the coupling influenced both the range and magnitude of the interaction force profiles between the silica colloid probe and the grafted PAAC layer; i.e., the concentration of EDC/NHS added during coupling of PAAC influenced the structure of the layer formed. This result was in good qualitative agreement with XPS analyses. The repulsive forces measured between the silica sphere and the PAAC layers exhibited a significant electrolyte concentration, pH, and molecular weight dependencies. For example, the magnitude and range of the interactions were constant over a pH range of 4.3-9.4. However, at a pH value of 3.1-3.3, the magnitude and range of the interaction were drastically reduced. The results obtained were compared to expectations based on theoretical studies and simulations, and good qualitative agreement was observed.

Introduction Immobilization of biologically active molecules (e.g., DNA, proteins, enzymes, peptides, drugs, etc.) onto solid supports is important for a wide variety of applications including biomaterials, tissue engineering, biosensors, immuno-assays, and DNA arrays. However, many polymer surfaces have undesirable surface properties that can lead to “failure” of the material and the device or system containing it. Examples of such failures may include (1) delamination of an adhesive bond, (2) fouling of membranes with proteinaceous films, (3) a false positive or false negative immuno-assay result, (4) bacterial growth on catheters, and (5) clotting of a dialyzer or blood oxygenator during dialysis or open-heart surgery.1 Covalent immobilization of biologically active molecules onto polymer surfaces can often lead to a significant reduction in the activity of the immobilized molecule. To avoid this problem, a spacer layer is often inserted between the substrate surface and the bioactive molecule, preferably one with low nonspecific interactions. For example, dextrans (e.g., carboxymethylated dextrans and partially amino-functionalized dextrans) have been used to immobilize bioactive molecules and/or to prevent protein adsorption.2-7 †

CSIRO Molecular Science. The University of New South Wales. § Current address: Department of Chemical Engineering, Intelligent Materials and Systems Institute, Universite´ de Sherbrooke, 2500, boul. Universite´, Sherbrooke, Que´bec, Canada, J1K 2R1. * To whom correspondence should be addressed: phone 61-39545-8121, Fax 61-3-9545-2446, e-mail [email protected]. ‡

(1) Biomaterials Science: An Introduction to Materials in Medicine; Ratner, B. D., Hoffman, A. S., Schoen, F. J., Lemons, J. E., Eds.; Academic Press: New York, 1996. (2) Brekkan, E.; Lundqvist, A.; Lundahl, P. Biochemistry 1996, 35, 12141. (3) Stachowiak, O.; Dolder, M.; Wallimann, T. Biochemistry 1996, 35, 15522.

In this study we investigate covalent immobilization of poly(acrylic acid) (PAAC) onto thin films deposited using radio-frequency glow discharge (RFGD) from a vapor of n-heptylamine (HApp) (as shown in Figure 1). Poly(acrylic acid), an anionic polyelectrolyte, is a valuable scaffold for immobilization of biologically active molecules because of the high density of carboxylic acid groups along its backbone, which may be used to covalently immobilize molecules containing amine groups, such as proteins. For example, we have used this scaffold to attach NeutrAvidin, a biotin binding protein.8,9 Poly(acrylic acid) graft layers have also been prepared using poly(olefin) and gold substrates for electrostatic incorporation of bioactive molecules and modification of surface properties.10,11 Development of suitable gel interlayers for subsequent immobilization of biologically active molecules requires a thorough understanding of the structure and chemistry of the solid-liquid interfacial region generated. In this study, characterization of the covalently grafted PAAC layers was carried out using surface-sensitive techniques including X-ray photoelectron spectroscopy (XPS) for determining surface composition and atomic force microscopy (AFM) in colloid probe force mode for probing the hydrated structure and thickness of the layers. Direct force measurements have been used extensively for (4) Piehler, J.; Brecht, A.; Hehl, K.; Gauglitz, G. Colloids Surf., B 1999, 13, 325. (5) McLean, K.; Johnson, G.; Chatelier, R.; Beumer, G.; Steele, J.; Griesser, H. Colloids Surf., B 2000, 18, 221. (6) McArthur, S.; McLean, K.; Kingshott, P.; St John, H.; Chatelier, R.; Griesser, H. Colloids Surf., B 2000, 17, 37. (7) Massia, S.; Stark, J.; Letbetter, D. Biomaterials 2000, 21, 2253. (8) Vermette, P.; Divisekera, U.; Gengenbach, T.; Griesser, H. J.; Meagher, L. Submitted to J. Colloid Interface Sci. (9) Vermette, P.; Meagher, L.; Gagnon, E.; Griesser, H. J.; Doillon, C. J. J. Controlled Release 2002, 80 (1-3), 179. (10) Franchina, J. G.; Lackowski, W. M.; Dermody, D. L.; Crooks, R. M.; Bergbreiter, D. E. Anal. Chem. 1999, 71, 3133. (11) Tao, G.; Gong, A.; Lu, J.; Sue, H.-J.; Bergbreiter, D. E. Macromolecules 2001, 34, 7672.

10.1021/la0202834 CCC: $22.00 © 2002 American Chemical Society Published on Web 12/02/2002

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Vermette and Meagher

Figure 1. Schematic diagram (not to scale) of the grafting of poly(acrylic acid) (PAAC) layers on n-heptylamine (HApp) thin films via water-soluble carbodiimide (EDC/NHS) chemistry.

investigating the structure of adsorbed neutral polymer and polyelectrolyte layers.12-22 The structures of adsorbed or grafted polymer layers are determined by the interactions of the polymer segments with each other, with the solvent molecules and ions present, and with the underlying surface.23 The structure of the surface layer may be probed by measuring the interaction forces between two of these layers or between one layer and a bare surface, as they are compressed. The range and magnitude of the interaction forces are determined, largely, by the grafted/adsorbed density of the polymer chains. To a first approximation, for purely repulsive forces at least, the range of the interaction forces indicates the thickness of the polymer layer. The results of this study not only provide thorough characterization of a suitable scaffold for subsequent immobilization reactions but also form the basis of a study of covalently grafted polyelectrolyte layers on solid surfaces. Experimental Section Materials. N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES) (99.5% purity), n-heptylamine (99% purity) used in plasma polymerization, poly(acrylic acid) (PAAC, 1200; 15, 30, 90, and 250 kDa), N-hydroxysuccinimide (NHS), and 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) were obtained from Sigma-Aldrich (Castle Hill, NSW, Australia). Sodium chloride (NaCl) (AnalaR) was obtained from Merck Pty. Limited (Kilsyth, Victoria, Australia). The 1 cm2 square silicon wafers were cut from 150 mm diameter wafer disks generously provided by Quality Semiconductor Australia (Homebush, NSW, Australia). Methods. Preparation of Buffer. The buffer used to determine the effect of the electrolyte concentration on the relationship between the silica colloid probe and the different surfaces contained 10 mM HEPES and 1, 10, or 100 mM NaCl and had (12) Dahlgren, M. A. G.; Waltermo, A.; Blomberg, E.; Claesson, P. M.; Sjostrom, L.; Akesson, T.; Jonsson, B. J. Phys. Chem. 1993, 97, 11769. (13) Dahlgren, M. A. G.; Claesson, P. M.; Audebert, R. J. Colloid Interface Sci. 1994, 166, 343. (14) Dahlgren, M. A. G.; Hollenberg, H. C. M. Ber. Bunsen-Ges. Phys. Chem. 1996, 100, 1004. (15) Luckham, P. F.; Hartley, P. G. Adv. Colloid Interface Sci. 1994, 49, 341. (16) Claesson, P. M.; Blomberg, E.; Paulson, O.; Malmsten, M. Colloids Surf. A: Physicochem. Eng. Aspects 1996, 112, 131. (17) Shubin, V. Langmuir 1994, 10, 1093. (18) Abe, T.; Higashi, N.; Niwa, M.; Kurihara, K. Langmuir 1999, 15, 7725. (19) Meagher, L.; Maurdev, G.; Gee, M. L. Langmuir 2002, 18, 2649. (20) Maurdev, G.; Meagher, L.; Ennis-King, J.; Gee, M. L. Macromolecules 2001, 34, 4151. (21) Muir, I.; Meagher, L.; Gee, M. Langmuir 2001, 17, 4932. (22) Hartley, P. G.; McArthur, S. L.; McLean, K. M.; Griesser, H. J. Langmuir 2002, 18, 2483. (23) Fleer, G. J.; Cohen Stuart, M. A.; Scheutjens, J. M. H. M.; Cosgrove, T.; Vincent, B. Polymers at Interfaces, 1st ed; Chapman and Hall: London, 1993.

a pH of 5.5. The solution used to determine the effect of the pH on the relationship between the silica colloid probe and the different surfaces contained 1 mM NaCl. Milli-Q gradient purified water (Millipore Australia Pty. Limited, North Ryde, NSW, Australia) with a resistivity of not less than 18.2 MΩ‚cm was used to prepare the buffer and NaCl solutions. Immobilization of Poly(acrylic acid). The surfaces were prepared using radio-frequency glow discharge (RFGD) techniques to deposit a cross-linked, organic thin film with amine functionality. Care was taken to use the RFGD films immediately to avoid oxidation in air.24-26 Deposition of thin RFGD polymer films was carried out from vapor of n-heptylamine in a custom-built reactor as described elsewhere.27 Briefly, the cylindrical reactor chamber is defined by a height of 35 cm and a diameter of 17 cm. Clean silicon wafer samples were placed on the lower circular electrode, which had a diameter of 9.5 cm. The upper electrode was U-shaped (length 17 cm, width 8 cm). The distance between the electrodes was 13 cm. The parameters chosen for the RFGD deposition of nheptylamine films were a frequency of 200 kHz, a load power of 20 W, and an initial monomer pressure of 17.33 Pa. Treatment time was 25 s. This results in a RFGD polymer film thickness of approximately 40 nm, as determined by AFM imaging.28 The root-mean-squared (rms) roughness of these RFGD polymer films was measured by AFM imaging and found to be approximately 0.3 nm over regions of several square micrometers.28 Poly(acrylic acid)s (PAAC) of different molecular weight were attached directly to n-heptylamine plasma polymer (HApp) using water-soluble carbodiimide chemistry.29 A 0.1% (w/v) solution of PAAC was prepared in Milli-Q water (pH adjusted to 3.5). Once dissolved, EDC and NHS were added at various concentrations (scenario 1: 1 mg cm-3 EDC + 0.1 mg cm-3 NHS; scenario 2: 19.2 mg cm-3 EDC + 11.5 mg cm-3 NHS; scenario 3: 0.1 mg cm-3 EDC + 0.1 mg cm-3 NHS), and the surfaces were immersed in the resultant solution. The reaction proceeded overnight at room temperature under vigorous shaking. To remove any noncovalently attached PAAC, the surfaces were rinsed overnight under vigorous shaking in a 1 M NaCl solution (the solution was changed twice), followed by rinsing in copious quantities of Milli-Q water. The surfaces were finally soaked overnight in Milli-Q water (the solution was changed twice) before being rinsed again in Milli-Q water and subsequently used. X-ray Photoelectron Spectroscopy. X-ray photoelectron spectroscopy (XPS) analysis was performed using an AXIS HSi spectrometer (Kratos Analytical Ltd., UK) equipped with a monochromated Al KR source at a power of 180 W. The pressure in the main vacuum chamber during analysis was typically 5 × 10-8 mbar. Elements present were identified from survey spectra. (24) Gengenbach, T. R.; Vasic, Z. R.; Chatelier, R. C.; Griesser, H. J. J. Polym. Sci., Part A: Polym. Chem. 1994, 32, 1399. (25) Gengenbach, T. R.; Chatelier, R. C.; Griesser, H. J. Surf. Interface Sci. 1996, 24, 271. (26) Gengenbach, T.; Vasic, Z.; Sheng, L.; Chatelier, R.; Griesser, H. Plasmas Polym. 1997, 2, 91. (27) Griesser, H. Vacuum 1989, 39, 485. (28) Hartley, P. G.; Thissen, H.; Vaithianathan, T.; Griesser, H. J. Plasmas Polym. 2000, 5, 47. (29) Hermanson, G. T. Bioconjugate Techniques; Academic Press: New York, 1996.

Poly(acrylic acid) Graft Layers

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Table 1. Elemental Compositions and Calculated Overlayer Thickness of Poly(acrylic acid) (PAAC) Grafted on n-Heptylamine Surfaces Prepared on Silicon Wafersa sample

%C

%O

%N

thickness (nm)

HApp HApp: PAAC(250 kDa MW), 1 mg cm-3 EDC + 0.1 mg cm-3 NHS HApp: PAAC(250 kDa MW), 19.2 mg cm-3 EDC + 11.5 mg cm-3 NHS HApp: PAAC(250 kDa MW), 0.1 mg cm-3 EDC + 0.1 mg cm-3 NHS HApp: PAAC (1200 MW) HApp: PAAC (15 kDa MW) HApp: PAAC (30 kDa MW) HApp: PAAC (90 kDa MW) HApp: PAAC (250 kDa MW)

76.6 69.9 74.2 75.4 72.4 73.8 73.5 71.8 69.9

17.6 26.8 20.7 19.7 23.2 21.5 22.1 24.6 26.8

5.9 3.3 5.1 4.9 4.3 4.7 4.4 3.5 3.3

0 1.9 0.4 0.6 1.0 0.7 0.9 1.6 1.9

a

The data were derived from carefully acquired XPS survey spectra.

High-resolution spectra were also collected at 40 eV pass energy (yielding a typical peak width for polymers of ca. 1 eV). Atomic concentrations of each element were calculated by determining the relevant integral peak intensities and applying the sensitivity factors supplied by the instrument manufacturer. A linear background was used in all cases. The random error associated with elemental quantification was determined to be 1-2% of the absolute values for atomic percentages in the range encountered in this study (>5 atom %).30 The systemic error was assumed to be of the order of 5-10%.24 A reference binding energy of 285.0 eV (aliphatic CHx) was used to correct for offsets due to charge neutralization of specimens under irradiation (typically ≈3.5 eV in this case). The electron attenuation length of a C 1s photoelectron in a polymeric matrix was assumed to be about 3 nm.24 This translates into an approximate value for the XPS analysis depth (from which 95% of the detected signal originates) of 10 nm when recording XPS data at an emission angle normal to the surface. A minimum of three positions on each sample were analyzed and the results averaged. Atomic Force Microscopy-Colloid Probe Force Measurements. The interaction forces between a silica particle and immobilized PAAC layers were measured with a Nanoscope multimode atomic force microscope (Digital Instruments, Inc., Santa Barbara, CA) using the colloid probe method developed by Ducker et al.31 In this method, a spherical colloidal particle was attached to the microfabricated AFM cantilever spring via an epoxy adhesive (Epon 1004, Resolution Performance Products, Houston, TX) and an XYZ translation stage, providing a surface of known geometry. In our case, the spherical particles were pure silica (diameter 4-5 µm), prepared by a modified Stober method,32,33 and were obtained from Bangs Laboratories, Inc. (Fishers, IN). To scale the force measurements correctly, the spring constant of the AFM cantilever must be known accurately. This was achieved using the resonance method proposed by Cleveland et al.34 This method gives the spring constant with an error of approximately 10%. An average value calculated from a sample of at least 10 cantilevers was used to scale the raw data obtained from the interaction force experiments. The cantilevers used were gold-coated, triangular Si3N4 cantilevers obtained from Digital Instruments Inc. (model NP) with spring constants of 0.076 and 0.155 N m-1. Conversion of the cantilever deflection curves to plots of the force/radius as a function of separation distance was carried out using a custom-designed computer program. The inputs for the program are specifications of zero force and separation distance as well as the radius of the spherical surface and the spring constant. In the analysis and scaling of the force profiles, the compliance or linear region of the deflection curve was used both to calibrate the photodetector and to define zero separation distance. The fittings and tubing used for injecting solutions into the AFM fluid cell were constructed from either Teflon or KelF polymers. These fittings, the fluid cell, O-ring, and syringe (used (30) Gengenbach, T. R. PhD, University of South Australia, 2000. (31) Ducker, W. A.; Senden, T. J.; Pashley, R. M. Nature (London) 1991, 353, 2239. (32) Stober, W.; Fink, A.; Bohn, E. J. Colloid Interface Sci. 1968, 26, 62. (33) Barder, T. J.; DuBois, P. D. US Patent No. 4,983,369, Jan 1991. (34) Cleveland, J. P.; Manne, S.; Bocek, D.; Hansma, P. K. Rev. Sci. Instrum. 1993, 64, 1.

for injecting solutions) were cleaned by placing the individual parts in a 1% surfactant solution (RBS-35, Pierce, Rockford, IL) overnight, followed by thorough rinsing with Milli-Q water and soaking twice in AR grade ethanol overnight using fresh ethanol each time. These components were blown-dry using a highvelocity stream of high-purity, compressed nitrogen. Solutions were prepared in glassware cleaned in the same manner but without the ethanol soaks. All operations were carried out in a laminar flow clean cabinet to minimize any particulate contamination. The silicon wafers were cleaned before RFGD deposition by immersion in a 1% RBS-35 surfactant solution overnight, rinsing thoroughly with Milli-Q water and finally blowing dry with a high-velocity nitrogen stream. The silicon wafers were then placed under a low-wavelength UV light in the presence of water vapor for 1 h to ensure that they were clean and completely hydrophilic.35 Then they were placed in the RFGD chamber for deposition of n-heptylamine thin films. Immediately before commencing an experiment, the AFM fluid cell and the AFM cantilever with silica sphere attached were placed under the UV light for 1 h. The average time between removing the cell and colloid probe from the UV lamp and injection of the first solution was 15 min. The general procedure used in AFM interaction force measurements was as follows. Freshly prepared silicon wafers containing immobilized PAAC and spherical silica surfaces were mounted into the AFM apparatus, brought to within a separation distance of 30 µm, a solution of HEPES buffer or NaCl was injected, and the surfaces were allowed to equilibrate for approximately 1 h. Force curves were then obtained and the solution conditions changed, followed always by equilibration for 1 h. At least 10 force curves were obtained for each sample and/or solution condition. The diameters of the spherical surfaces were measured using a video camera and screen attached to a high-power optical microscope (final magnification 2460×).

Results and Discussion XPS analysis of HApp thin films on the silicon wafers indicated a polymer rich in hydrocarbon- and nitrogencontaining species (see Table 1 and Figure 2). The broad C 1s peak (see Figure 2) is associated with a variety of chemical structures, formed during thin film deposition from the gas plasma. As a result, it was difficult to clearly resolve the C-N-containing species from those containing C-O, which may result from the spontaneous quenching of carbon radicals within the film on exposure to air. Previous studies using derivatization have, however, indicated that these coatings have an amine density of approximately 0.5-2.0 amines/nm2.36 The reduction in silicon atomic concentration from ∼37% for an uncoated oxidized silicon wafer (data not shown) to zero when coated also indicated a >10 nm thick, pinhole-free RFGD polymer layer. This has been confirmed by AFM imaging, which showed films prepared in an identical manner to be 3540 nm in thickness.28 (35) Vig, J. R. J. Vac. Sci. Technol. A 1985, 3, 1027. (36) Griesser, H.; Chatelier, R. J. Appl. Polym. Sci.: Appl. Polym. Symp. 1990, 46, 361.

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Figure 2. High-resolution XPS C 1s spectra of HApp surface and HApp surface bearing covalently grafted PAAC (90 kDa, 1 mg cm-3 EDC + 0.1 mg cm-3 NHS).

XPS analysis of the HApp thin film modified silicon wafer samples following EDC/NHS grafting of PAAC demonstrated a significant increase in oxygen content relative to the HApp surface, which confirmed the attachment of poly(acrylic acid) (PAAC) (Table 1). It should be noted that the control HApp surfaces were treated in exactly the same manner as the samples with coupled PAAC; thus, the degree of oxidation should be the same in all samples. To compare the high-resolution C 1s peak positions, the spectra were shifted to ensure that the leading edges of the fitted aliphatic CHx component were coincident. All spectral intensities were normalized to a maximal intensity corresponding to the full height of the fitted aliphatic CHx (285.0 eV) component peak. Typical high-resolution C 1s XPS spectra obtained from a HApp and a grafted PAAC surface (90 kDa molecular weight, 1 mg cm-3 EDC + 0.1 mg cm-3 NHS) were compared (Figure 2). Of particular note was the intensity increase in the peak at approximately 289 eV relative to the plasma polymer, which is indicative of carboxyl incorporation at the surface. Comparison to published XPS reference spectra37 verified that the high-resolution C 1s spectra were typical of PAAC. The presence of PAAC was not detected by XPS when EDC/NHS were omitted during the coupling of PAAC to HApp. The percent oxygen in the PAAC surfaces was dependent on the coupling conditions used (i.e., the concentrations of EDC and NHS) (Table 1). The highest percent oxygen was obtained using 1 mg cm-3 EDC + 0.1 mg cm-3 NHS. This effect was reflected clearly in the O/C and N/C atomic percent ratios (Figure 3A). The highest O/C and the lowest N/C ratios were obtained when 1 mg cm-3 EDC + 0.1 mg cm-3 NHS were used in the coupling reaction. As N was only present in the HApp substrate, the lowest N/C represents the thickest PAAC coating. Overlayer thickness calculations using a simple model38 were also carried out (Table 1). The value taken for the inelastic mean free path of an electron with a binding energy of 404 eV was 3.25 nm.39 The calculation was carried out assuming that the overlayer of PAAC was continuous, of uniform thickness/grafting density, and that an ejected photoelectron behaves in a similar manner in a grafted layer as in a bulk polymer. The calculated thickness varied from 0.4 to 1.9 nm as the coupling conditions were varied. This calculation assumes that the (37) Beamson, G.; Briggs, D. High-Resolution XPS of Organic Polymers: The Scienta ESCA300 Database; John Wiley and Sons: Chichester, 1992. (38) Tielsch, B. J.; Fulghum, J. E. Surf. Interface Sci. 1994, 21, 621. (39) Tanuma, S.; Powell, C. J.; Penn, D. R. Surf. Interface Anal. 1993, 21, 165.

Figure 3. O/C or N/C ratios derived from quantification of XPS survey spectra of poly(acrylic acid) (PAAC) layers grafted on n-heptylamine surfaces using different (A) various EDC/ NHS coupling conditions and (B) different molecular weights of PAAC. The error bars denote the range of values obtained from at least three positions on each sample.

density of the coupled layer was constant. Hence, any decrease in N atomic percent was reflected as an increase in thickness. Equally likely was an increase in PAAC coupling density (coupled chains per unit area). Whether the grafted layer was thicker or denser in a vacuum, the end result of this series of experiments was an optimized mass per unit area for a given set of coupling conditions. These optimized coupling conditions (i.e., 1 mg cm-3 EDC and 0.1 mg cm-3 NHS), which resulted in the thickest PAAC layers, were used for all subsequent experiments except for AFM experiments with samples of varying PAAC grafting density (see below). There have been other studies of EDC/NHS coupling conditions.40,41 These studies found that the coupling density was a complex function of pH, [EDC]/[NHS], and concentrations of the carboxylic acid, amine surface density, and the buffer used. However, these studies used quite disparate coupling conditions (the pH in particular). Our study used a low pH (as in the Pieper40 study) and obtained an optimized [EDC]/[NHS] ratio that was similar to that obtained by Stile et al.41 Clearly, this is an area in need of further study. In our minds there are other parameters that are of importances for example, the conformation of the polymer molecule in solution and the ratio of [EDC] to the concentration of carboxylic acid-containing segments of the polymer chains, in relation to the amine surface site density. This last parameter should have the greatest impact on the structure of the coupled layer since the thickest structure will be that in which only a small percentage of the acid (40) Pieper, J.; Hafmans, T.; Veerkamp, J.; van Kuppevelt, T. Biomaterials 2000, 21, 581. (41) Stile, R. A.; Barber, T. A.; Castner, D. G.; Healy, K. E. J. Biomed. Mater. Res. 2002, 61, 391.


groups react with the surface amines. A possible interpretation of these data is that the lowest concentrations of EDC and NHS (0.1/0.1 mg/mL) result in little activation of the PAAC molecules in solution and therefore inefficient coupling and the lowest coupled mass. The moderate concentration values (1.0/0.1 mg/mL) result still in incomplete activation of the PAAC molecules in solution but enough to give a grafted layer that contains much more mass and with a conformation containing a significant proportion of loops and tails. Certainly, the surface force profiles suggest that this is indeed the case. Higher EDC and NHS concentrations could result in efficient activation but with the result that the grafted conformation is much flatter and less mass is grafted due to unavailability of surface sites. Thus, there is a maximum in grafted mass as a function of EDC and NHS concentration. An analogy may be made with adsorbed polymer layers where there is a strong attractive interaction potential between the surface and the segments of the chains, low adsorbed amounts, and flat conformations, compared to less attractive interactions, where the conformation contains more loops and tails and larger adsorbed amounts are obtained. As the molecular weight of PAAC was increased, more PAAC was coupled to the surface (with one exception, 1.2 kDa) (Figures 1 and 2). This general result was reflected in the increases in % O (Table 1) and the O/C ratio (Figure 3B), decreases in % N (Table 1), and the N/C ratio (Figure 3B) and an increase in the calculated thickness of the PAAC layer (Table 1) as the molecular weight of the PAAC was increased. In addition, no apparent differences were noted in the high-resolution C 1s spectra between the grafted PAAC layers having different molecular weights (data not shown). Assuming the same density of coupling sites in each case, it makes intuitive sense that higher molecular weights would result in thicker and/or denser layers. The objective of the study was to produce a layer with a maximum number of possible COOH coupling sites for further reaction (for example, with proteins). Thus, only two molecular weights were studied in any further detail. XPS analysis of the grafted PAAC layers provided a measure of the composition and approximate thickness in a vacuum. However, for biomedical applications it is mandatory to characterize the surfaces in aqueous environment, particularly the thickness, structure, and chemical functionality of the coatings. We used the AFM colloid probe method for the direct measurement of interaction forces between a silica probe and the grafted PAAC surfaces as our primary characterization tool. As mentioned previously, the specific conditions used to couple the PAAC to the HApp substrate resulted in varying grafting densities. The same surfaces were characterized by AFM in order to assess the impact of the EDC/NHS coupling conditions on the grafting density and therefore on the thickness and structure of the coatings in various ionic strength salt solutions. However, it is important to give an impression of the baseline force data obtained previously in this laboratory22 for interacting silica and HApp surfaces without any grafted PAAC. Briefly, when measurements between silica and HApp were carried out in solutions of low electrolyte concentration (effective concentration of electrolyte of 1.5 mM PBS, pH 7.2), the force/radius vs separation relationships obtained were purely repulsive in nature and decayed exponentially. The decay length measured in this study was ∼7.5 nm, which is in reasonable agreement with the theoretical decay length for a force of electrostatic origin for surfaces immersed in an electrolyte solution of this

Langmuir, Vol. 18, No. 26, 2002 10141

Figure 4. Normalized forces (approach traces) between a silica sphere and n-heptylamine polymer surfaces bearing poly(acrylic acid) immersed in buffer solutions containing 10 mM HEPES and 1.0, 10, and 100 mM NaCl (pH 5.5). The surfaces were prepared using grafting solutions containing (A) 1 mg cm-3 EDC + 0.1 mg cm-3 NHS, (B) 19.2 mg cm-3 EDC + 11.5 mg cm-3 NHS, and (C) 0.1 mg cm-3 EDC + 0.1 mg cm-3 NHS.

concentration (calculated theoretical κ-1 ) 7.8 nm). The repulsive, electrostatic nature of the measured force indicates that at the experimental pH both surfaces were negatively charged. When the electrolyte concentration was increased to pure PBS (phosphate buffered saline, effective 1:1 electrolyte concentration ) 150 mM), the electrostatic repulsive forces were removed completely, and there was no interaction between the surfaces prior to hard wall contact. The measured interaction forces between a silica colloid probe and HApp surface bearing poly(acrylic acid) (250 kDa MW) grafted using different EDC/NHS conditions were plotted as log F/R vs separation distance, a convention that clearly identifies force profiles that decay exponentially (Figure 4). The force profiles were repulsive, roughly

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exponentially decaying, and of long range. However, the decay lengths of the force profiles presented do not match the theoretically calculated value for purely electrostatic interactions between surfaces immersed in salt solutions of the concentration used here. For example, the interaction forces in 0.001 M NaCl displayed a decay length of approximately 35 nm compared to a value of 9.6 nm for a purely electrostatic force (Figure 4A). The ramifications of the differences in decay lengths of the various forces may be illustrated by comparing the range of interaction forces for two charged surfaces immersed in a 0.001 M solution of 1:1 electrolyte (on the order of 30-40 nm). Here we see measured forces that extend to well over 200 nm in a 0.001 M NaCl solution. Thus, the interactions measured for the PAAC surfaces were not purely electrostatic in nature and cannot be described by the DLVO theory. We can conclude from this that the interaction force profiles presented here are largely a result of compression of the covalently attached PAAC layers by the silica sphere. The concentration of EDC/NHS added during the coupling influenced both the range and magnitude of the interaction force profiles between the silica colloid probe and the grafted PAAC layers (Figure 4). Comparison of these data indicates that the concentration of the EDC/ NHS used to covalently attach the PAAC onto the HApp surfaces influences the structure (e.g., thickness, conformation, and coverage) of the grafted poly(acrylic acid) layers. For example, when 1 mg cm-3 EDC and 0.1 mg cm-3 NHS (Figure 4A) were used to covalently attach the PAAC molecules onto the HApp surfaces, the resulting interaction forces were of greater magnitude and longer range than any other set of EDC/NHS concentrations. We interpret this to mean that the PAAC grafted layer was thicker (more extended toward the solution) than when, for example, 0.1 mg cm-3 EDC and 0.1 mg cm-3 NHS (Figure 4C) were used. In this case we define the thickness of the layer to be the maximum range of the interaction forces between the layer and the silica particle. That is, the grafting density was much higher under these coupling conditions. From consideration of the results obtained from molecular dynamics simulations42 and self-consistent-field theoretical calculations,43,44 there should be a direct relationship between the grafted layer thickness and the grafting density of the layer. Unfortunately, it is difficult to calculate the grafting density of PAAC due to the polydisperse nature of the polyelectrolyte molecules, the fact that several grafting points per molecule are likely, and the significant errors associated with estimating the mass per unit area from the XPS analysis. However, there is good qualitative agreement between the theoretical predictions, the interaction force results, and the XPS analyses (Figure 3A). The assumption made in presenting this interaction force data is that the error in defining zero separation distance is relatively small so that comparisons of force curves obtained under different conditions can be reliably made and are not misleading. In the constant compliance region, the PAAC layer is highly compressed by the silica particle, such that the slope of the spring deflection vs piezo travel data in this region appears to be constant; i.e., the layer is behaving as a noncompressible solid. In addition, the thicknesses estimated for these layers in ultrahigh vacuum via XPS overlayer calculations were on (42) Csajka, F. S.; Seidel, C. Macromolecules 2000, 33, 2728. (43) Zhulina, E. B.; Borisov, O. V.; Birshtein, T. M. J. Phys. II 1992, 2, 63. (44) Zhulina, E. B.; Wolterink, J. K.; Borisov, O. V. Macromolecules 2000, 33, 4945.


the order of 1-2 nm thickness. An extensive survey of the interaction force data for adsorbed or grafted polymer systems presented in the literature12-14,17,20,45-70 indicates that the compressed thickness of the covalently attached PAAC layer would most likely be on the order of 5-10 nm and possibly somewhat smaller. Clearly, the error in thickness (estimated from the range of the interaction forces) is dependent on the range of the interaction forces. In most cases presented in Figures 4-6, approximately exponentially decaying, repulsive forces were observed between the silica colloid probe and the polymer surface in all electrolyte conditions. A smooth transition was observed between the exponential behavior, shorter range, nonexponential repulsive forces and hard wall repulsion (designated as to zero separation). However, the form of these force profiles does not indicate that the forces are purely electrostatic in origin like those for HApp surfaces.22 While there is an electrostatic component, it is the electrostatic interaction between the negatively charged carboxylic acid groups along the polymer chain backbones that results in the repulsive forces observed here. Compression of the chains by the silica particle on approach forces these negatively charged groups closer together on average. Since like charges repel each other, the chains resist compression resulting in a repulsive force. In addition, the local density of the polymer segments is increased on compression. While it is clear that these forces should indeed be repulsive, it is not necessarily obvious that the form should be exponential. The structure of isolated, grafted polyelectrolyte layers has been extensively studied in the past 20 years using theory and simulation. The early work carried out by Fleer and co-workers23,71 has been greatly expanded by Zhulina (45) Abe, T.; Kunihara, K.; Higashi, N.; Niwa, M. J. Phys. Chem. 1995, 99, 1820. (46) Abraham, T.; Giasson, S.; Gohy, J. F.; Jerome, R. Langmuir 2000, 16, 4286. (47) Ananthapadmanabhan, K. P.; Mao, G.-Z.; Goddard, E. D.; Tirrell, M. Colloids Surf. 1991, 61, 167. (48) Argillier, J.-F.; Ramachandran, R.; Harris, W. C.; Tirrell, M. J. Colloid Interface Sci. 1991, 146, 242. (49) Claesson, P. M.; Dedinaite, A.; Blomberg, E.; Sergeyev, V. G. Ber. Bunsen-Ges. Phys. Chem. 1996, 100, 1008. (50) Claesson, P. M.; Paulson, O. E. H.; Blomberg, E.; Burns, N. L. Colloids Surf. A: Physicochem. Eng. Aspects 1997, 123-124, 341. (51) Claesson, P. M.; Fielden, M. L.; Dedinaite, A.; Brown, W.; Fundin, J. J. Phys. Chem. B 1998, 102, 1270. (52) Dahlgren, M. A. G.; Claesson, P. M. Prog. Colloid Polym. Sci. 1993, 93, 206. (53) Dahlgren, M. A. G. Langmuir 1994, 10, 1580. (54) Dedinaite, A.; Claesson, P. M.; Bergstrom, M. Langmuir 2000, 16, 5257. (55) Dedinaite, A.; Claesson, P. M. Langmuir 2000, 16, 1951. (56) Fielden, M. L.; Claesson, P. M.; Schillen, K. Langmuir 1998, 14, 5366. (57) Hadziioannou, G.; Patel, S.; Granick, S.; Tirrell, M. J. Am. Chem. Soc. 1986, 108, 2869. (58) Israelachvili, J. N.; Tirrell, M.; Klein, J.; Almog, Y. Macromolecules 1984, 17, 204. (59) Kjellin, U. R. M.; Claesson, P. M.; Audebert, R. J. Colloid Interface Sci. 1997, 190, 476. (60) Le Berre, F.; Malmsten, M.; Blomberg, E. Langmuir 2001, 17, 699. (61) Luckham, P. F.; Klein, J. J. Chem. Soc., Faraday Trans. 1 1984, 80, 865. (62) Malmsten, M.; Claesson, P. M.; Pezron, E.; Pezron, I. Langmuir 1990, 6, 1572. (63) Marra, J.; Hair, M. J. Phys. Chem. 1988, 92, 6044. (64) Osterberg, M.; Laine, J.; Stenius, P.; Kumpulainen, A.; Claesson, P. M. J. Colloid Interface Sci. 2001, 242, 59. (65) Pelletier, E.; Stamouli, A.; Belder, G. F.; Hadziioannou, G. Langmuir 1997, 13, 1884. (66) Pezron, I.; Pezron, E.; Claesson, P. M.; Malmsten, M. Langmuir 1991, 7, 2248. (67) Rojas, O. J.; Claesson, P. M.; Muller, D.; Neuman, R. D. J. Colloid Interface Sci. 1998, 205, 77. (68) Sukhishvili, S. A.; Dhinojwala, A.; Granick, S. Langmuir 1999, 15, 8474.


and co-workers43,44 and others.72,73 The shape of the segment density profile of the polyelectrolyte brush was found to be exponentially decaying or Gaussian in shape in low electrolyte concentrations. This segment density profile should be reflected in the interaction force profiles. The most likely outcome of the polydispersity introduced into the grafted layers investigated here (multiple pinning points, polydisperse PAAC) would be that the segment density profile would become even more exponential in nature. Another finding of the theoretical studies was that higher salt concentrations result in a more parabolic segment density profile, a result that will be discussed in more detail layer. In addition to studies of isolated layers, the interaction of grafted and adsorbed polyelectrolyte layers has also been theoretically examined.74-76 In some instances the results indicate that the interactions of two similarly charged grafted layers are strongly repulsive and exponential in nature,74,77 similar to the experimental results presented here. The effect of salt concentration on the range and magnitude of these forces has also been examined theoretically and will be compared with the results obtained for our grafted PAAC layers below. In the data presented here, there was no indication of a long-range electrostatic interaction between the negatively charged PAAC layer and the silica surface as has been observed in other studies.78 In particular, this effect was observed in cases where the polyelectrolyte was adsorbed from low ionic strength salt solution, resulting in a fairly flat conformation. The lack of a long-range electrostatic tail has also been observed in studies of two adsorbed polyelectrolyte layers, where the adsorbed amount was very high. In this case the force profiles were also exponentially decaying.79 Given the assumed segment density distribution for the layers here, the charge present at the extremities of the layer would be small and therefore any electrostatic force would be small also, perhaps below the sensitivity of the AFM force technique. The repulsive forces presented in Figures 4 and 5 also exhibited a significant electrolyte dependency. That is, the range and magnitude of the interaction forces were reduced as the bathing electrolyte concentration was increased. While this behavior appears analogous to the electrical double-layer compression observed for the unmodified plasma polymer surfaces, comparison of the measured vs theoretical decay lengths for 1:1 electrolyte systems reveals considerable discrepancies. In addition, as the salt concentration was increased, the reduction in the magnitude and range decreased so that for the 250 kDa PAAC layer (see Figure 5A) the differences between 10 and 100 mM were small compared to the differences between 1 and 10 mM. Therefore, the changes in the force profiles are a result of electrolyte screening of the charges along the polymer backbone. As the electrolyte concentra(69) Taunton, H. J.; Toprakcioglu, C.; Fetters, L.; Klein, J. Macromolecules 1990, 23, 571. (70) Tirrell, M.; Patel, S.; Hadziioannou, G. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 4725. (71) Fleer, G. J. Ber. Bunsen-Ges. Phys. Chem. 1996, 100, 936. (72) Dan, N.; Tirrell, M. Macromolecules 1993, 26, 4310. (73) Seidel, C. Macromolecules 1994, 27, 7085. (74) Pincus, P. Macromolecules 1991, 24, 2912. (75) Israels, R.; Leermakers, F. A. M.; Fleer, G. J. Macromolecules 1995, 28, 1626. (76) Miklavic, S. J.; Woodward, C. E.; Johsson, B.; Akesson, T. Macromolecules 1990, 23, 4149. (77) Sjostrom, L.; Akesson, T.; Jonsson, B. J. Chem. Phys. 1993, 99, 4739. (78) Kamiyama, Y.; Israelachvili, J. N. Macromolecules 1992, 25, 5081. (79) Maurdev, G.; Gee, M. L.; Meagher, L. Langmuir, in press.

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Figure 5. Normalized forces (approach traces) between a silica sphere and n-heptylamine polymer surfaces bearing poly(acrylic acid) immersed in buffer solutions containing 10 mM HEPES and 1.0, 10, and 100 mM NaCl (pH 5.5). The surfaces were prepared using grafting solutions containing 1 mg cm-3 EDC + 0.1 mg cm-3 NHS and (A) 250 kDa MW and (B) 90 kDa MW PAAC.

tion was increased, the repulsive interaction between the polymer charges was reduced, resulting in less stretching of the chains away from the surface, into the solution. There is considerable experimental evidence in support of this finding. For example, Guo and Ballauff80 observed, via dynamic light scattering, that the thickness of a grafted poly(acrylic acid) brush decreased as the electrolyte concentration was increased. Similar effects have been observed for poly(styrenesulfonate)81 and polybase82 brushes. These results are supported by theoretical calculations that show that the range and magnitude of repulsive pressures between two grafted polyelectrolyte layers were reduced by adding salt.74 Thus, as the bathing salt concentration is increased, the polymers chains behave more and more like neutral polymers in a good solvent. However, there are some theoretical results that suggest that a transition from good to poor solvency conditions occurs for polyelectrolyte chains, which accompanies increases in electrolyte concentration, due to counterion condensation.42 In addition, the theoretical studies of isolated polyelectrolyte brushes44 find also that the thickness depends markedly on the electrolyte concentra(80) Guo, X.; Ballauff, M. Langmuir 2000, 16, 8719. (81) Ahrens, H.; Forster, S.; Helm, C. A. Macromolecules 1997, 30, 8447. (82) Wesley, R. D.; Cosgrove, T.; Thompson, L.; Armes, S. P.; Billingham, N. C.; Baines, F. L. Langmuir 2000, 16, 4467.

10144


tion. In particular, a transition to a parabolic segment density distribution at high salt concentration is noted. This would manifest as parabolic force profiles as has been observed many times for monodisperse neutral brushes.83,84 Clearly, for the results presented in Figure 5, the polydispersity of the brushes studied here most likely precludes a parabolic segment density profile. Also apparent in the data presented in Figure 5A,B was a significant molecular weight dependence of the range and magnitude of the interaction forces between silica and the PAAC layers. This effect is supported by the results of molecular dynamics simulations for grafted polyelectrolyte layers42 and by scaling theory.72,85 The simulations predict a linear dependence between layer thickness and number of segments in the polymer chains. Note that this simulation was for end-grafted chains whereas the system under study here most likely has several grafting points per molecule. Nonetheless, the same trend might be expected. As only two molecular weights were studied, these data are not sufficient to establish a relationship between thickness and molecular weight for these PAAC layers. In addition, it is not clear how the addition of salt would influence this relationship, since many of the theoretical studies have been carried out with the assumption of no added salt. The interaction force data for PAAC layers coupled using optimal EDC (1 mg cm-3) and NHS (0.1 mg cm-3) concentrations for a molecular weight of 250 and 90 kDa in 0.001 M NaCl solutions as a function of pH were measured to examine the effect of varying the charge density of the grafted polyelectrolyte on the interaction forces (Figure 6). The magnitude and range of the interactions were constant (within experimental error) over a pH range of 4.3-9.4. However, at a pH value of 3.1-3.3, the magnitude and range of the interactions were drastically reduced. Thus, the driving force for stretching the polymer chains into solution (i.e., the repulsive interactions between the charged segments along the polymer backbones) was reduced due to protonation of the carboxylic acid groups. The reported pKa value for PAAC in solution is 4.5,86 which suggests that the polyelectrolyte is strongly dissociated over a wide range of pH values, as would be expected for carboxylic acid groups,87 and the general trends in dissociation agree quite well with the force data presented here. However, titrations of PAAC molecules show that the molecules are not fully dissociated until pH 8 and that the degree of dissociation varies in a smooth fashion between pH 3 and 8. This behavior is very different from that obtained here for the range of interaction forces vs solution pH. The difference can probably be explained by counterion condensation, measurements of which indicate that the effective charge is constant after pH 6.888 and drops sharply below pH 5.0. Interestingly, at low pH values, the polymer layers collapse to a thickness of approximately 50 nm for both molecular weights studied. This value may be compared to the collapse of the layers in 0.1 M NaCl to thickness values of approximately 130 and 40 nm for the 250 and (83) Leckband, D.; Sheth, S.; Halperin, A. J. Biomater. Sci., Polym. Ed. 1999, 10, 1125. (84) Efremova, N. V.; Bondurant, B.; O’Brien, D. F.; Leckband, D. E. Biochemistry 2000, 39, 3441. (85) Israels, R.; Leermakers, F. A. M.; Fleer, G. J.; Zhulina, E. B. Macromolecules 1994, 27, 3249. (86) Anghel, D.; Alderson, V.; Winnik, F.; Mizusaki, M.; Morishima, Y. Polymer 1998, 39, 3035. (87) Mandel, M. Eur. Polym. J. 1970, 6, 807. (88) Boisvert, J. P.; Malgat, A.; Pochard, I.; Daneault, C. Polymer 2002, 43, 141.


Figure 6. Normalized forces (approach traces) between a silica sphere and n-heptylamine polymer surfaces bearing poly(acrylic acid) immersed in 1 mM solutions of NaCl at various pH values. The surfaces were prepared using grafting solutions containing 1 mg cm-3 EDC + 0.1 mg cm-3 NHS and (A) 250 kDa MW and (B) 90 kDa MW PAAC.

90 kDa PAAC, respectively. Useful comparisons may be made between the data obtained here and that obtained by Hartley et al.,22 who measured the streaming potential of covalently grafted, partially oxidized (50%) carboxymethyldextran (CMD) coatings on HApp surfaces. These authors found that the negative potential associated with the grafted layer approached zero at a pH of approximately 3.4. While the magnitude of the potential measured is not strictly accurate for extended polyelectrolyte layers such as CMD, due to the diffuse nature of the region of surface charge and uncertainty as to the location of the shear plane, the data point clearly to the pH region where most of the carboxyl groups on the CMD are protonated. While it is not clear what influence covalent grafting and the proximity of a high concentration of carboxylic acid groups has on the dissociation behavior of PAAC molecules, it is apparent from the data in Figure 6 that there is a very large difference between the dissociation of the grafted PAAC molecules between pH values of 4.3 and 3.3. Complete protonation of the acid groups most likely occurs below pH 3.3-3.1. However, it was not possible to reduce the pH further and maintain a constant solution ionic strength. Another comparison may be drawn between the data in Figure 6 and interaction force data obtained for self-assembled monolayers (SAMs) of alkanethiols terminated with carboxylic acid groups.89 These carboxylic (89) Hu, K.; Bard, A. J. Langmuir 1997, 13, 5114.


acid groups appear to have substantially different dissociation behavior, apparently fully protonated at pH 6, with an apparent pKa of approximately 8. This behavior is very different from that obtained here, for grafted CMD surfaces, and for PAAC molecules in solution and may relate to the high density of surface groups obtained with SAMs. Other favorable comparisons may be made between the data presented in Figure 6 and that obtained for other grafted acid layers by other methods. For example, optically detected pH titrations of polyacid brushes,90,91 dynamic light scattering,80 and ellipsometry.91,92 In addition, the results obtained here are in qualitative agreement with theoretical predictions based on variations in linear charge density.43,85 Conclusions Successful immobilization of PAAC onto HApp was demonstrated in this study by XPS analyses and AFM colloid probe force measurements. The force profiles obtained between silica particles and the PAAC coating were repulsive in nature, roughly exponentially decaying and of long range. Comparison of the decay lengths obtained with those expected for purely electrostatic interactions indicated that the repulsive forces were due to electrosteric interactions. That is, there was an electrostatic component of the steric force generated as the polyelectrolyte chains were compressed by the silica particle. The concentration of EDC/NHS added during (90) Currie, E. P. K.; Sieval, A. B.; Avena, M.; Zuilhof, H.; Sudholter, E. J. R.; Stuart, M. A. C. Langmuir 1999, 15, 7116. (91) Currie, E. P. K.; Sieval, A. B.; Fleer, G. J.; Stuart, M. A. C. Langmuir 2000, 16, 8324. (92) Biesalski, M.; Ruhe, J.; Johannsmann, D. J. Chem. Phys. 1999, 111, 7029.

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the coupling influenced both the range and magnitude of the interaction force profiles between the silica colloid probe and the grafted PAAC layer. This was in good qualitative agreement with XPS analyses. This indicated that the concentration of the EDC/NHS used to covalently attach the PAAC onto the HApp influenced the structure and density of the grafted PAAC layers. In addition, the repulsive forces measured between the silica sphere and the PAAC layers exhibited a significant electrolyte and molecular weight dependency with respect to the range and magnitude. That is, the range and magnitude of the interaction forces were reduced both as the electrolyte concentration was increased and as the molecular weight of the PAAC was reduced from 250 to 90 kDa. It was also shown that the magnitude and range of the interaction were constant over a pH range of 4.3-9.4. However, at a pH value of 3.1-3.3, the magnitude and range of the interaction were drastically reduced. The results of this study not only provide thorough characterization of a suitable scaffold for subsequent immobilization reactions (e.g., of biologically active molecules) but also form the basis of a study of covalently grafted polyelectrolyte layers. The behavior exhibited by the grafted PAAC layers was in very good qualitative agreement with theoretical studies and simulations of grafted polyelectrolyte layers. Acknowledgment. We thank Hans J. Griesser from CSIRO Molecular Science for his helpful guidance and collaboration. This research at the Cooperative Research Centre for Eye Research and Technology was partially supported by the Australian Federal Government under the Cooperative Research Centres Scheme (P.V., L.M.) and by the FCAR-Que´bec (P.V.). LA0202834

Immobilization and Characterization of Poly(acrylic acid) - American

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