In-Situ Investigation of the Adsorption of Globular Model Proteins on

Dec 8, 2006 - ... versatile tool for surface engineering to tune physicochemical surface characteristics such as wettability, surface charge, chemical...
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Langmuir 2007, 23, 57-64

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In-Situ Investigation of the Adsorption of Globular Model Proteins on Stimuli-Responsive Binary Polyelectrolyte Brushes† Petra Uhlmann,*,‡ Nikolay Houbenov,‡,| Nina Brenner,‡,§ Karina Grundke,‡ Sina Burkert,‡ and Manfred Stamm‡ Leibniz Institute of Polymer Research Dresden, Dresden, Germany, UniVersity of Helsinki, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Helsinki, Finland, and Department of Engineering Physics and Mathematics, Optics and Molecular Materials Group, Helsinki UniVersity of Technology, HUT, Espoo, Finland ReceiVed May 31, 2006. In Final Form: NoVember 3, 2006 Binary brushes constituted from two incompatible polymers can be used in the form of ultrathin polymeric layers as a versatile tool for surface engineering to tune physicochemical surface characteristics such as wettability, surface charge, chemical composition, and morphology and furthermore to create responsive surface properties. Mixed brushes of oppositely charged weak polyelectrolytes represent a special case of responding surfaces that are sensitive to changes in the pH value of the aqueous environment and therefore represent interesting tools for biosurface engineering. The polyelectrolyte brushes used for this study were composed of two oppositely charged polyelelctrolytes poly(2-vinylpyridine) (P2VP) and poly(acrylic acid) (PAA). The in-situ properties and surface characteristics such as as surface charge, surface tension, and extent of swelling of these brush layers are functions of the pH value of the surrounding aqueous solution. To test the behavior of the mixed polylelctrolyte brushes in contact with biosystems, protein adsorption experiments with globular model proteins were performed at different pH values and salt concentrations (confinement of counterions) of the buffer solutions. The influence of the pH value, buffer salt concentration, and isoelectric points (IEP) of the brush and protein on the adsorbed amount and the interfacial tension during protein adsorption as well as the protein adsorption mechanism postulated in reference to recently developed theories of protein adsorption on polyelectrolyte brushes is discussed. In the salted regime, protein adsorption was found to be similar to the often-described adsorption at hydrophobic surfaces. However, in the osmotic regime the balance of electrostatic repulsion and a strong entropic driving force, “counterion release”, was found to be the main influence on protein adsorption.

1. Introduction A very promising means to optimize surface properties is the deposition of thin polymeric layers. Grafting techniques (i.e., the covalent linkage of the polymer to a surface) have several advantages such as easy and controllable introduction of polymer chains with high surface density, precise localization of the chain at the surface, the possibility of grafting different polymers on the same substrate, and the long-term stability of the grafted layers. The term “brush” in this context means a dense layer of chainlike polymers with one end fixed on a substrate and the mean grafting distance shorter than the mean size of the polymers.1 When two (or more) incompatible polymers are grafted, the anchoring of the polymer chains prevents macroscopic separation of the system, allowing the creation of surface properties that on one hand combine the properties of the grafted polymers and on the other hand allow switching of the surface characteristics. Therefore, mixed polymer brushes created from two or more polymers represent polymer systems with remarkably responsive properties (i.e., they are able to adapt and respond to external fields and environmental conditions). The phase behavior of mixed polymer brushes is determined by the competition of the mixing entropy, which favors the homogeneously mixed state, † Part of the Stimuli-Responsive Materials: Polymers, Colloids, and Multicomponent Systems special issue. * To whom correspondence should be addressed. E-mail: uhlmannp@ ipfdd.de. Tel: +49-351-4658-236. Fax: +49-351-4658-284. ‡ Leibniz Institute of Polymer Research Dresden. § University of Helsinki. | Helsinki University of Technology.

(1) Milner, S. T. Science 1991, 251, 905.

with the interaction energy, which is reduced by spatial separation of the incompatible polymers.2 Binary systems of mixed polymer brushes can be used, for example, to create surfaces that respond to environmental stimuli by switching between (ultra)hydrophilic and (ultra)hydrophobic or rough to smooth surface properties.3-8 Polymer brushes can be fixed on inorganic substrates (such as SiO2 surfaces3,4) as well as on polymer surfaces as a polyamide.5-8 Flat surfaces can be modified just as particles or powders.9,10 Binary brushes were also used to create reversible environment-responsive surface patterning by photocrosslinking one of the components11,12 and obtaining gradient layers for combinatorial approaches.13-17 (2) Marko, J. F.; Witten, T. A. Phys. ReV. Lett. 1991, 66, 1541-1544. (3) Minko, S.; Luzinov, I.; Luchnikov, V.; Mu¨ller, M., Patil, S.; Stamm, M. Macromolecules 2003, 36, 7268-7279. (4) Sidorenko, A.; Minko, S.; Schenk-Meuser, K.; Duschner, H.; Stamm, M. Langmuir 1999, 15, 8349 (5) Minko, S.; Mu¨ller M.; Motornov, M.; Nitschke, M.; Grundke, K. Stamm, M. J. Am. Chem. Soc. 2003, 125, 3896-3900. (6) Grundke, K.; Nitschke, M.; Minko, S.; Stamm, M.; Froeck, C.; Simon, F.; Uhlmann, S.; Po¨schel, K.; Motornov, M. Contact Angle, Wettability Adhes. 2003, 3, 1-25. (7) Motornov, M.; Minko, S.; Nitschke, M.; Grundke, K.; Stamm, M. Polym. Prepr. 2002, 43, 379. (8) Motornov, M.; Minko, S.; Eichhorn, K. J.; Nitschke, M.; Simon, F.; Stamm, M. Langmuir 2003, 19, 8077-8085. (9) Sidorenko, A.; Minko, S.; Gafijchuk, G.; Voronov, S. Macromolecules 1999, 32, 4539-4543. (10) Motornov, M.; Stamm, M.; Minko, S. Polym. Prepr. 2005, 46(2), 78. (11) Ionov, L.; Minko, S.; Stamm, M.; Gohy, J. F.; Jerome, R.; Scholl, A. J. Am. Chem. Soc. 2003, 125, 8302-8306. (12) Ionov, L.; Stamm, M.; Minko S.; Hoffmann, F.; Wolff, T. Macromol. Symp. 2004, 207, 229-236. (13) Zdyrko, B.; Kleb, V.; Luzinov, I.; Minko, S.; Sydorenko, A.; Ionov, L.; Stamm M. Polym. Prepr. 2003, 44, 522.

10.1021/la061557g CCC: $37.00 © 2007 American Chemical Society Published on Web 12/08/2006

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Such systems also represent a suitable concept to tune surface and interfacial properties of biomaterials. The primary processes that are occurring when a material surface comes into contact with a biological organism or biofluid is the interaction with proteins and cells. These processes involve the modification of the physical and chemical properties of the interface on the one hand and changes in the structure and properties of the involved proteins and cells on the other hand, which causes a systemic response of the organism to contact with the material. A variety of attempts have been made during the last few decades to control the biological response to a material surface (e.g., by physicochemical surface modification or immobilization of biomolecules). Besides the integral surface properties, micro- and nanoscaling interfacial properties, dynamics, and the ability to adapt to external stimuli are more and more regarded as important in the design of biomaterial interfaces18. Thus, certain ratios of polar and apolar surface functionalities19 were proposed, which was corroborated by proposing the concept of a microdomain structure.20 Because polymer brushes represent such systems having microor nanodomains (e.g., polar and apolar, oppositely charged) coupled with a special conformation of the polymer chains (brushlike), they offer a promising and versatile tool for use in modifying biomaterials in this sense and in controlling the physicochemical constraints for the adsorption of bioactive molecules at the interface. Weak polyelectrolyte brushes appear to be especially promising for such purposes, as a special case of responsive surfaces, because the properties of such layers in an aqueous environment can be tuned by influencing the electrostatic interactions and surface hydrophobicity/hydrophilicity with pH21 and the osmotic pressure of the counter ions with added salt, which is important for interactions with biomacromolecules as proteins and cells. Studies investigating the driving forces of protein adsorption have demonstrated the importance of enthalpic contributions as van der Waals, electrical double layer, and hydrophobic interactions. However, entropically based mechanisms are also important. They comprise the release of counter ions and/or water of solvation, the structural reorganization of water molecules due to protein adsorption (belonging to hydrophobic interactions), and the reduction of ordered structures due to adsorption-induced conformational changes.22-24 The importance of entropic driving forces was recently shown by investigating the adsorption of model proteins on spherical polyelecrolyte brushes25-30 and also (14) Ionov, L.; Houbenov, N.; Sidorenko, A.; Minko, S.; Stamm, M. Polym. Mater. Sci. Eng. 2004, 90, 104. (15) Ionov, L.; Houbenov, N.; Sidorenko, A.; Minko, S.; Stamm, M. Macromol. Rapid Commun. 2004, 25, 360-365. (16) Ionov, L.; Houbenov, N.; Sydorenko, A.; Stamm, M.; Luzinov, I.; Minko, S. Langmuir 2004, 20, 9976-9979. (17) Ionov, L.; Sidorenko, A.; Stamm, M.; Minko, S.; Zdyrko, B.; Kleb, V.; Luzinov, I. Macromolecules 2004, 37, 7421-7423. (18) Galaev, I. Y.; Mattiasso,n B. Trends Biotechnol. 1999, 17, 335. (19) Hanson, S. R.; Harker, L. A.; Ratner, B. D.; Hoffman, A. S. Ann. Biomed. Eng. 1979, 7, 357-367. (20) Goldberg, E. P., Nakajima, A., Eds. Polymeric Materials and Pharmaceuticals for Biomedical Use; Academic Press: New York, 1980. (21) Houbenov, N.; Minko, S.; Stamm, M. Macromolecules 2003, 36, 58975901. (22) Norde, W. In Biopolymers at Interfaces; Malmsten, M.. Ed.; Surfactant Science Series; Marcel Dekker: New York, 1998; Vol. 75. (23) Norde, W. Colloids and Surfaces in Life Sciences; Marcel Dekker: New York, 2003. (24) Malmsten, M. J. Colloid Interface Sci. 1998, 207, 186-199. (25) Anikin, K.; Ro¨cker, C.; Wittemann, A.; Wiedenmann, J.; Ballauff, M.; Nienhaus, U. J. Phys. Chem. B 2005, 109, 5418. (26) Czeslik, C.; Jansen, R.; Ballauff, M.; Wittemann, A.; Royer, C. A.; Gratton, E.; Hazlett, L. Phys. ReV. E 2004, 69, 021401. (27) Jackler, G.; Wittemann, A.; Ballauff, M. Spectroscopy 2004, 18, 1671. (28) Rosenfeldt, S.; Wittemann, A.; Ballauff, M.; Breininger, E.; Bolze, J.; Dingenouts, N. Phys. ReV. E 2004, 70, 061403.

Uhlmann et al.

through a microcalorimetric investigation of the formation of protein/polyelectrolyte complexes.31 Polyelctrolyte brushes are differentiated into strong and weak ones. The degree of dissociation of weak polyelectrolytes (annealed brush) is dependent on the pH value of the surrounding solution, whereas strong polyelectrolytes are fully dissociated and strongly charged species (quenched brush).32,33 Scaling laws for the thickness of the brushes and their interactions were developed by Pincus34 using a theoretical treatment. The thickness of a polyelectrolyte brush was shown to result from a balance of the osmotic pressure of the counterions inside the brush and the configurational elasticity of the chains. Derived from that, one can define two states of polylelctrolyte brushes: in the osmotic regime (i.e., at low concentrations of additional ions, where the ion concentration inside the brush is higher than it is outside), the majority of the counterions condense inside the brush. The osmotic pressure dominates, and the chains may be stretched to almost full length. The thickness L of the brushes is determined by the degree of dissociation f and the chain length (number of segments N × length of a segment a) but is independent of the grafting density.

L ≈ f1/2Na

(1)

Weakly charged polyelectrolytes with a low grafting density represent a special case. In this case, a portion of the counterions are situated outside the brush, and the brush is behaving similarly to a strongly charged surface.35 The thickness of the brush then also depends on the Bjerrum length. If the ion concentration of the surrounding solution is at least as high as the ion concentration inside the brush, then the brush is in the so-called salted regime. The electrostatic forces within the brush are highly screened, the osmotic pressure of the counterions is disappearing, and the brush is shrinking. In this case, the thickness of the brush is a function of the external salt concentration cs and the grafting density Fa:

L ≈ Na(2acs)-1/3Fa1/3

(2)

In an extended theory, attractive interactions due to electrostatic correlations are included.36 Depending on the chain length and grafting density, complex phase behavior is postulated, leading to two additional brush regimes: the quasi-neutral brush and the collapsed brush. Theoretical considerations by Linse et al. showed that mixed brushes of oppositely charged polymers show rather complicated behavior due to interactions between the two charged polymers influencing the chain conformation.37 This leads to a complex response to an increase in the charge density along the chain and to salt addition, which may differ from that of homopolyelectrolyte brushes. The experiments to prove the other were not done until now, whereas experiments to prove the other above-mentioned theories were performed in different groups,38-39 and most intensively (29) Wittemann, A.; Haupt, B.; Ballauff, M. Phys. Chem. Chem. Phys. 2003, 5, 1671. (30) Wittemann A.; Ballauff M. Macromol. Biosci. 2005, 111, 225. (31) Ball, V.; Winterhalter, M.; Schwinte, P.; Lavalle, Ph.; Voegel, J. C.; Schaaf, P. J. Phys. Chem. B 2002, 106, 2357-2364. (32) Guo, X.; Ballauff, M. Phys. ReV. E 2001, 64, 051406-1-8. (33) Biesalski, M.; Johannsmann, D.; Ruehe, J. J. Chem. Phys. 2002, 117, 10, 4988-4994. (34) Pincus, P. Macromolecules 1991, 24, 2912. (35) Deserno, M.; Holm, C.; May, S. Macromolecules 2000, 33, 199. (36) Csajka, F. S.; Netz, R. R.; Seidel, C.; Joanny, J. F. Eur. Phys. J. E 2001, 4, 505. (37) Shusharina, N. P.; and Linse, P. Eur. Phys. J. E 2001, 6, 147-155. (38) Balastre, M.; Li, F.; Scorr, P.; Yang, J.; Mays, J. W.; Tirrell, M. V. Macromolecules 2002, 35, 9480.

Adsorption of Proteins on Polyelectrolyte Brushes

for spherical polyelectrolyte homobrushes in Ballauff’s group by using scattering and spectroscopic methods.32,40-44 Because we were interested in adaptive or switchable surfaces, we used binary brushes of weak polyelectrolytes (i.e., annealed brushes). These brushes were composed of the two oppositely charged weak polyelelctrolytes poly(2-vinylpyridine) (P2VP) and poly(acrylic acid) (PAA). The properties of these brushes are described in detail in ref 21 and will be summarized here: These mixed poylelctrolyte brushes show a sharp switching of the surface charge, which depends on the pH of the environmental solution. The isoelectric point (IEP), determined from streaming potential measurements in 0.001 mol/L KCl solution, was found to be 4.9. Therefore, the mixed brushes show amphiphilic behavior (i.e., the surface can be both negatively and positively charged as a function of pH). At pH 3.2 (IEP ) 3.221) it is more and more negatively charged as a result of the increasing dissociation of the carboxylic surface groups with increasing pH). In the region of 3.2 < pH < 6.7, the charged P2VP and PAA chains interact, resulting in an uncharged surface at pH 4.9 (cf. ref 21). This behavior could be illustrated by investigating the swelling of the polylecrolyte mixed brush layers at different pH values by in-situ ellipsometry in a batch cell. Minimal swelling that corresponded to the thickness of the dry layer (6.4 nm) was found near the IEP. At pH values above and below the IEP, a sharp increase in swelling was observed as a result of the highly protonated P2VP chains at low pH values and the deprotonated PAA chains at high pH values, respectively. Therefore, it is possible to switch the surface charge from negative to positive and to create even uncharged surfaces near the IEP of the mixed brush. For these changes, only small pH differences are necessary. This was also confirmed by contact angle measurements where a maximum was found near the IEP. The contact angles for high and low pH values were found to be similarly low, which is proof of the symmetry of the binary polyelectrolyte brushes. 2. Experimental Section Materials. Carboxy-terminated poly(tert-butyl acrylate) (PBACOOH, Mn ) 42 000, Mw ) 47 000 g/mol) and poly(2-vinylpyridine) (P2VP-COOH, Mn ) 39 000, Mw ) 41 500 g/mol) were both obtained from Polymer Source Inc., Canada, 3-glycidoxypropyltrimethoxysilan (GPS) was obtained from Aldrich, and silicon wafers (orientation 100) with ca. 1.5 nm native SiO2 were obtained from Wacker Chemtronics. Cleaning of Silicon Wafers. The wafers were treated several times with dichloromethane in an ultrasonic bath and then exposed to the cleaning solution (NH4OH/H2O2/water ) 1:1:1) for 2 h at 60 °C. Silanization was performed using 1% GPS solution in toluene for 16 h. Grafting of the Polymers. The so-called grafting-to procedure was used (i.e., the polymers were linked to the surface from polymer solution via a coupling layer (GPS) exhibiting reactive groups (epoxy) that are likely to bind covalently to the carboxy-terminated polymers). The mixed brushes were synthesized using a two-step mechanism: A thin layer of carboxy-terminated PBA was deposited on the GPS (39) Hayashi, S.; Abe, T.; Higashi, N.; Niwa, M.; Kurihara, K. Langmuir 2002, 8, 3932. (40) Robillard, Q.; Guo, X.; Dingenouts, N.; Ballauff, M.; Goerigk, G. Macromol. Symp. 2001, 164, 81. (41) Mei, Y.; Sharma, G.; Lu, Y.; Ballauff, M.; Drechsler, M.; Irrgang, T.; Kempe, R. Langmuir 2005, 21, 12229. (42) Mei, Y.; Ballauff, M. Eur. Phys. J. E 2005, 16, 341. (43) Dingenouts, N.; Ballauff, M.; Pontoni, D.; Narayanan, T.; Goerigk, G. Macromolecules 2004, 37, 8152. (44) Jusufi, A.; Likos, C. N.; Ballauff, M. Colloid Polym. Sci. 2004, 282910.

Langmuir, Vol. 23, No. 1, 2007 59 Table 1. Properties of the Globular Model Proteins45,46 protein

molecular weight (Da)

R-chymotrypsin

25 200

R-lactalbumin

14 200

size (nm)

IEP (pH units)

secondary structure

5.1 × 4 × 4

8.1

3.7 × 3.2 × 2.5

4.3

11% R-helix, 51% β-sheet 26% R-helix, 14% β-sheet

layer via spin coating from a 1% methanol solution and annealed in a vacuum oven at 150 °C for 20 min. The noncovalently bound PBA was removed by Soxhlet extraction with methanol. The second polymer P2VP-COOH was spin coated in the same way and annealed for 15 h. After Soxhlet extraction with THF, PBA was converted into poly(acrylic acid) (PAA) by treatment with a benzene solution saturated with p-toluene sulfonic acid monohydrate at 55 °C for 1 h. The grafting kinetics were controlled in such a way that symmetrical brushes were generated. The resulting brushes had a grafting density of 0.1 nm-2 and a distance between the grafting points of 3 nm (details in ref 21). Proteins. The properties of the used model proteins are summarized in Table 1. R-Chymotrypsin (type II, bovine pancreas, C-4129) and R-lactalbumin (bovine milk, L-5385) were obtained from Sigma-Aldrich. The proteins were dissolved in phosphate-buffered saline (PBS, Sigma-Aldrich, Germany) containing 0.01 M phosphate buffer, 0.0027 M KCl, 0.137 M NaCl, and 0.01 M Tris or citrate buffer (prepared from citric acid monohydrate, both Sigma-Aldrich, Germany). The pH values were adjusted by the addition of 1 N NaOH or HCl. These buffers were also described as high-salt solutions in the case of PBS and low-salt solutions in the case of Tris and citrate buffer. The protein concentration used was 0.25 mg/mL.

3. Methods In-Situ Streaming Ellipsometry. A single-wavelength ellipsometer (ELX-02C, Dr. Riss Ellipsometerbau GmbH, Ratzeburg, Germany) was equipped with a He-Ne laser (wavelength 632.8 nm). The self-made experimental setup that was used for the streaming experiments consisted of a flow cell (glass cuvette, open on both sides, with an angle of incidence of 68°, TSL spectrosil B, Hellma, Mu¨llheim, Germany) that was connected to a constant-pressure pumping system. To adjust the liquid flow circulating over the sample, gas-pressure pumps (operating by adjusting a defined gas pressure over the liquid to be pumped) were used to pump the liquid constantly from one vessel to another so that the direction of flow over the sample was not changing. For one measurement, 60-90 mL of protein solution and 200 mL of buffer solution are needed. The fluid flow is adjusted by regulating the inlet gas pressure (nitrogen 5.0) via the voltage control of a power supply. A disadvantage of the setup was that relatively high volumes of protein solution were needed. The setup and pumping system will be described in detail in ref 47. Such a setup makes it possible to create a constant flow over the sample surface (wall shear rate ) 7.8 s-1) with a homogeneous concentration distribution. Because the solutions can be switched from one reservoir to the other, it is possible to perform continuous measurements of adsorption, desorption, and displacement of (bio)macromolecules. In-Situ Batch Ellipsometry. All measurements were carried out using a null ellipsometer (Multiscope, Optrel Berlin) in polarizer-compensator-analyzer mode also operating at a wavelength of 632.8 nm and an angle of incidence of 68° and equipped with a batch cuvette (TSL Spectrosil, Hellma, Muellheim, Germany). After equilibration of the polymer brush in the buffer solution for 40 min, protein solution was injected into the cuvette and carefully mixed to reach an overall protein concentration in the cuvette of 0.25 mg/mL. After finishing the adsorption

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Figure 1. Principal setup of the contact angle experiment using ADSA-P, which was performed to investigate protein adsorption in-situ.

experiment, the solution in the cuvette was changed back to pure pH-adjusted buffer solution or to buffer solutions of a different pH to investigate the switching by pH. The desorption measurements were carried out for approximately 40 min. For the evaluation of the protein adsorption experiments and the determination of the adsorption layer thicknesses on polylectrolyte brushes, an optical four-layer model (silicon substrate with n ) 3.8705-j 0.0168, SiO2 and GPS as an effective optical layer with n ) 1.445, the brush as an effective optical layer with n calculated according to the composition of the brush (PVP with n ) 1.5037 and PAA with n ) 1.527),21 protein with n ) 1.3318,48 with n-refractive index) was used. In-Situ ADSA-P Measurements. ADSA (axisymmetric drop shape analysis) is a technique to determine solid-liquid interfacial tensions and contact angles from the shape of sessile (or pendant) drops.49 Assuming that the experimental drop is Laplacian and axisymmetric, the strategy employed by ADSA is to fit the shape of an experimental drop profile according to the Laplace equation of capillarity using a least-square algorithm with the surface (interfacial) tension as an adjustable parameter. The best fit identifies the correct surface or interfacial tension from which the contact angle can be determined by a numerical integration of the Laplace equation. Besides the contact angle, the drop volume, surface area, and three-phase contact radius can also be computed simultaneously. Details are given in ref 50. The experimental drop profile is acquired using an automatic digitization technique with subpixel resolution. In our previous work, the surface state of solid-liquid interfaces was studied before, after, and during the adsorption of proteins by using a special “captive bubble” technique.51 Here another in-situ contact angle technique was applied to monitor simultaneously the changes in the contact angle and the liquid surface tension from which the solid-liquid interfacial tension could be calculated:52-55 A droplet of the buffer solution with a volume of 95 µL was deposited on the polymer surface and allowed to equilibrate. Then 5 µL of a more concentrated protein solution was added by using a micropipet to reach a final protein concentration in the droplet of 0.25 mg/mL. A principal scheme of the experiment is shown in Figure 1. Using ADSA-P, it is possible to compute simultaneously changes in the liquid surface tension γlv, the contact angle θ, and contact radius r that are caused by the adsorption of the added protein at the interface. (45) Arai, T.; Norde, W. Colloids Surf. 1990, 19, 1-15. (46) Norde, W., Zoungrana, T. Biotechnol. Appl. Biochem. 1998, 28, 133143. (47) Uhlmann, P.; Ko¨rber, H.; Werner, C. To be submitted to Colloids Surf., B. (48) Werner, C.; Eichhorn, K. J.; Grundke, K.; Simon, F.; Gra¨hlert, W.; Jacobasch, H. J. Colloids Surf. 1999, 156, 3-17. (49) Rotenberg, Y. R.; Boruvka, L.; Neumann, A. W. Colloid Interface Sci. 1983, 93, 169. (50) Lahooti, S.; del Rio O. I.; Cheng, P.; Neumann, A. W. In Applied Surface Thermodynamics; Neumann, A. W., Spelt, K., Eds.; Marcel Dekker: New York, 1996; p 441. (51) Grundke, K.; Werner, C.; Po¨schel, K.; Jacobasch, H. J. Colloids Surf. 1999, 156, 19. (52) Noordmans, J.; Wormeester, H.; Busscher, H. J. Colloids Surf., B 1999, 15, 227.

Figure 2. Scheme of the net surface charges of the interacting proteins and the brush dependent on their IEP and the pH value of the buffer solution during the performed experiments.

To reduce the evaporation of the droplet, the experiments were performed in a glass cuvette. The drop volume V can also be recorded during the experiment. From the experimental parameters θ(t) and γlv(t), changes in the solid-liquid interfacial tension γsl(t) can be computed at every moment of the experiment using Young’s equation

γsl(t) ) γsv - γlv(t) cos θ(t)

(3)

assuming that the solid surface tension γsv at the solid-gas interface is not changing during the adsorption experiment. γsv can be determined separately from the advancing contact angle of water on the corresponding polymer surface and was determined to be 47.83 mN/m.

4. Results and Discussion The sketch in Figure 2 gives an overview of the experimental conditions concerning the chosen globular proteins and the net surface charges of interacting proteins and brushes during the performed experiments. Adsorbed Amount of Protein. The adsorbed amount was calculated from the ellipsometrically measured layer thickness by using a frequently used equation that was originally proposed by de Fejter56

Γ ) dp

(

)

np - nl dn/dc

(4)

in which dp is the layer thickness of the adsorbed protein layer, np is the refractive index of the protein layer, nl is the refractive index of the ambient solution, and dn/dc is the refractive index increment. This method uses the assumption that the refractive index of a protein in solution is a linear function of the protein concentration. dn/dc was set to 0.18757,58 for both proteins. The experiments were performed with R-chymotrypsin and R-lactalbumin at pH values of 4, 6, and 9 with (high salt) and without additional salt (low salt). A typical experiment is shown in Figure 3. The experiments were performed in three steps: (1) equilibration of the brush in buffer solution, (2) protein adsorption, and (3) rinsing with buffer solution. First, we investigated whether the use of streaming or batch ellipsometry (i.e., an applied flow of liquid through the cuvette) has an influence on the adsorbed amount. The principle of the (53) Van der Vegt, W.; Norde, W.; Van der Mei, H. C.; Busscher, H. J. Colloid Polym. Sci. 1996, 274, 27. (54) Van der Vegt, W.; Van der Mei, H. C.; Busscher, H. J. J. Colloid Interface Sci. 1993, 156, 129. (55) Van der Vegt, W.; Van der Mei, H. C.; Busscher, H. J. Langmuir 1994, 10, 13. (56) de Fejter, J. A.; Benjamins, J.; Veer, F. A. Biopolymers 1978, 17, 1759. (57) Malmsten, M. J. Colloid Interface Sci. 1994, 166, 333-342. (58) Malmsten, M.; Lassen, B. J. Colloid Interface Sci. 1994, 166, 490-498.

Adsorption of Proteins on Polyelectrolyte Brushes

Figure 3. Adsorbed amount of protein during a typical experiment of the adsorption of R-chymotrypsin on a mixed polyelctrolyte brush (pH 9, low salt).

Figure 4. Amount of R-chymotrypsin (plateau values) adsorbed from different buffer solutions, obtained by streaming ellipsometry.

ellipsometric setup and the dimensions of the cuvette were the same in both cases. The applied wall shear rate of 7.8 s-1 (which is quite low) obviously had little influence on the adsorbed amount of the proteins (cf. Figures 4 and 5a) and the adsorption mechanisms were not changed; only the reproducibility of the measurements was slightly better in the case of streaming ellipsometry experiments, which was certainly due to the more homogeneous concentration distribution in solution and at the interface. From these experiments, it was concluded that the less material- and time-consuming batch ellipsometry will give the same benefit when investigating the adsorption mechanism of proteins on brushes, hence it was decided to use only batch ellipsometry for subsequent experiments. The results obtained for the two investigated model proteins are summarized in Figure 5. It is immediately to be seen that big differences between high and low salt solutions and of the behavior of the two investigated proteins were found. R-Chymotrypsin. In low-salt buffer, the adsorbed amount increased considerably with pH. An influence neither of electrostatic interactions (repulsion at pH 4 and 9, attraction at pH 6) nor of the hydrophilic-hydrophobic balance (maximum hydrophobicity at pH 6) of the brush was seen. The very high adsorbed amount at pH 9 is conspicuous. In high-salt buffer, the electrostatic interactions are screened, and the adsorbed amounts decreased in comparison to those in the low-salt solutions. Much smaller differences were found at pH 4 and 6 compared to that at pH 9 (cf. Figure 5a). In Figure 5c, the protein adsorption at a PAA-P2VP mixed brush is compared with that at a PtBA (poly(tert-butylacrylate)-P2VP mixed brush. The PtBA-P2VP brush (having no carboxylic groups) was adsorbing much less protein compared to the PAA-P2VP brush in the low-salt buffer but almost the same amount as that in the high-salt buffer.

Langmuir, Vol. 23, No. 1, 2007 61

R-Lactalbumin. A completely different behavior was observed when adsorbing R-lactalbumin from the same buffers. From Figure 5b, can be seen that the adsorbed amount was decreasing with increasing pH when being adsorbed from high-salt as well as low-salt buffers. Increasing the salt concentration decreased the adsorbed amount at all investigated pH values. Almost no differences were found in the adsorption capacity of PtBA or PAA containing mixed brushes. The adsorbed amounts were very low in both cases (Figure 5d). In-Situ Wetting Experiments. These experiments were performed with R-chymotrypsin to determine if interfacial processes were involved in the considerable increase in the adsorbed amount with pH in low-salt buffer. In Figure 6, a typical ADSA-P experiment is shown. After about 800 s, the protein solution was injected into the droplet of the buffer solution, which caused a significant and fast decrease in the liquid surface tension of the droplet (γlv(t)), a decrease in the contact angle (θ(t)), and an increase in the contact radius (r(t)) (i.e., a spreading of the droplet). Immediately after adding the protein to the buffer solution droplet, the contact angle increases before it starts to decrease. It is assumed that this behavior is caused by the pinning of the triple line while the droplet volume is increased. The changes in interfacial tension (γsl(t)) were calculated from eq 1 using γsv values that were calculated from contact angle measurements with water. The results are summarized in Table 2. It can be seen that during the protein adsorption from solution with low ionic strengths the contact angle decrease caused by protein injection was more pronounced than from solutions with high ionic strengths. In the case of low salt, these differences were increasing with pH as the adsorbed amounts measured by ellipsometry did, but the increase in the adsorbed amount was much larger. In the high-salt buffer, the most pronounced decrease in the contact angle was found at pH 6 (i.e., where the brush has its maximal hydrophobicity). The interfacial tensions reached their initial value after a relaxation time of almost 2 h in the case of low-salt buffers at pH 4 and 6 and decreased in buffers at pH 9. Because in low-salt buffers the brush is in the osmotic regime (i.e., the chains are stretched), interactions of the protein molecule with the brush causing conformational changes and rearrangement processes of both interacting partners should be responsible for the slow relaxation of the interfacial tension. However, only in buffers at pH 9 is a new equilibrium reached, which is manifested as a decrease in interfacial tension. In high-salt buffers (i.e., when the protein is interacting with a shrunken or even collapsed brush (salted regime)), the decrease in interfacial tension after relaxation is more pronounced. Here it is very probable that changes in the protein layer adsorbing on the outside of the brush are responsible for the decrease in interfacial tension. It is well known that on hydrophobic surfaces proteins are most likely to change their conformation, and it was also found that on those surfaces changes in the interfacial tension (due to rearrangement processes and conformational changes of the protein) are still going on after the plateau value of adsorbed amount is reached.22 This was also found here. Regarding this, it is understandable why the most pronounced decrease in interfacial tension was found for the adsorption of R-chymotrypsin from a buffer of pH 6. Adsorption Mechanism in Low-Salt Buffer. The brush is in the osmotic regime under these conditions (i.e., the chains are expanded or stretched, and the ion concentration (counterions) inside the brush is much larger than in the surrounding medium). This behavior is schematically shown in Figure 7 for low and high pH values. R-Chymotrypsin behaved very differently than R-lactalbumin during adsorption under these conditions.

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Figure 5. Amounts of R-chymotrypsin and R-lactalbumin (plateau values) adsorbed from different buffer solutions, obtained by batch ellipsometry: adsorption of R-chymotrypsin (a) and R-lactalbumin (b) on a PAA-P2VP mixed brush; comparison of the adsorption of R-chymotrypsin (c) and R-lactalbumin (d) on a PAA-P2VP and a PtBA-P2VP mixed brush.

Figure 6. Typical in-situ ADSA-P experiments for the adsorption of R-chymotrypsin on a polylectrolyte brush. Simultaneous recording of θ(t), γlv(t), r(t), and γsl(t): low-salt buffer, pH 4, without changing γsl during adsorption (left) and high-salt buffer, pH 6, with a decrease in γsl due to adsorption.

R-Chymotrypsin was very strongly adsorbed from a low-salt buffer at pH 9 although the adsorption took place under repulsive conditions (i.e., on the wrong side). Hence, steric and electrostatic repulsions had to be overcompensated by another force that leads to strong adsorption. Because R-chymotrypsin was described as a “hard” (i.e., conformationally stable) protein,45,46 entropically based contributions due to adsorption-induced conformational changes are not likely on this hydrophilic surface. Biesheuvel59-61 et al. were theoretically describing (using the Boublik(59) Biesheuvel, P. M.; Leermakers, F. A. M.; Cohen Stuart, M. A. Phys. ReV. E 2006, 73, 011802. (60) Biesheuvel, P. M.; Wittemann, A. J. Phys. Chem. B 2005, 109, 4209.

Mansoori-Carnahan-Starling-Lelang excess function for multicomponent mixtures of spheres) how a negatively charged globular protein adsorbs in a negatively charged PAA brush. They concluded that such adsorption on the “wrong side” is possible when the ionic strength is low enough and the pH is not too much above the IEP of the protein. According to this theory, the protein is regarded as amphoteric, and upon entry into the brush, its charge reverses. Ion-release driving forces and an increase in charge of the PAA chains are postulated.1 It was calculated that ion release is the largest contributor to protein (61) Biesheuvel, P. M.; van der Vees, M.; Norde, W. J. Phys. Chem. B 2005, 109, 4172-4180.

Adsorption of Proteins on Polyelectrolyte Brushes

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Table 2. Adsorption of r-Chymotrypsina ∆θ, deg

γsl(0) [mJ/m2]

γsl (e) [mJ/m2]

pH

θ0, deg

θe, deg

4 6 9

55.6 61 50.6

38.4 42 26.7

low salt 17.3 19 23.8

9 9 7

9 9 3

4 6 9

54.6 58 52.2

44.6 41.8 38

high salt 10 16.2 14

9 9 9

9 2 5

a Results of the ADSA experiments: θ , contact angle before injection 0 of the protein; θe, equilibrium contact angle at the end of the experiment; ∆θ, contact angle difference caused by protein adsorption; γsl(0), interfacial tension before protein injection; and γsl (e), interfacial tension at the end of the experiment. Average relative error is 5%.

Figure 7. Scheme of the interaction of a globular protein with a mixed PAA-P2VP brush in low-salt buffer solution at pH 4 (left) when the brush is exposing slightly positive charges to the interface and at pH 9 (right) with the brush exposing expanded negatively charged PAA chains to the interface. At pH 9, the steric conditions for the interacting protein are different compared to those at pH 4 (P2VP chains are much less stretched).

adsorption (∆Fion entropy ) -59.9kT) whereas the increase in brush ionization provides a contribution of ∆Fchem brush ) -24.4kT (i.e., about 30% of the total driving force). The importance of the entropic contribution due to counterion release with protein/ polyelectrolyte interactions was also proven by Ball et al. through microcalorimetry experiments investigating the formation of protein/ polyelectrolyte complexes being, for their system, an endothermic process.31 Wittemann and Ballauff et al.25-30 showed in a variety of protein adsorption experiments on spherical PAA brushes that counterion release is a very strong entropic driving force for protein adsorption because the proteins acting as multivalent counterions (as a result of the inhomogeneous charge distribution on the surface, i.e., “patchiness”) of the brush release a large number of confined small counterions from the brush. They also showed that proteins can enter the spherical PAA brushes. We postulate that the adsorption of R-chymotrypsin on the mixed polyelectrolyte brush on a planar surface follows the same mechanism. Theoretically postulated differences in the complex response of a mixed brush compared to those of a homobrush obviously do not influence the adsorption mechanism. The reason for the decreasing adsorbed amount of R-lactalbumin with increasing pH is the increasing electrostatic repulsion with increasing pH due to the very close IEP of the protein and the brush. The repulsion is obviously strong enough that R-lactalbumin is not able to enter the brush and to act as a multivalent counterion of the brush (i.e., in this case, there is no entropic driving force due to counterion release). However, R-lactalbumin is known as a “soft” protein62,63 and is able to adsorb on the “wrong” side by being able to compensate for the electrostatic repulsion forces by entropically based contributions due to adsorption-induced conformational changes.

Figure 8. Scheme of the interaction of a globular protein with a mixed PAA-P2VP brush in a high-salt buffer solution.

Adsorption Mechanism in High-Salt Buffer. The brush is in the salted regime (ca > 0.1 M) (i.e., the polymer chains are partially collapsed, see Figure 8). Electrostatic interactions between proteins and the brush are screened. Because of the partial collapse of the brush, the proteins are repelled by steric interactions. This is why the adsorbed amount of protein generally decreases when adding additional salt. In the case of R-chymotrypsin, added salt decreased the adsorbed amount considerably and all the more as the counterion release force contributes to adsorption. When R-lactalbumin is adsorbed, additional salt concentration generally decreases the adsorbed amount, a behavior that is well known for the adsorption of proteins on polyelectrolytes because of molecule shrinkage. The higher adsorbed amount of R-lactalbumin compared to that of R-chymotrypsin at pH 4 is certainly due to the higher conformational flexibility of R-lactalbumin leading to higher entropically based contributions (due to protein unfolding) to the driving force, which is very likely because the shrunken brush and the hydrophobic surface behave similarly. Reversibility of Protein Adsorption by Changing the pH Value in Low-Salt Buffer. The experiments were performed by adsorbing the model proteins at pH 4 or 9. After the plateau was reached, the protein solution was exchanged by a buffer solution with a different pH (9 or 4) without offering additional protein to be adsorbed during the second step of the experiment. When performing the experiments in this manner, the surface charges of the protein and brush and the brush conformation will change depending on pH. From Figure 9 it can be seen that the adsorbed R-chymotrypsin at pH 9 can be removed by changing the pH of the buffer to pH 4 (Figure 9a). This is not the case when adsorbing R-chymotrypsin from a buffer at pH 4 (Figure 9b) and was also not found for the adsorption of R-lactalbumin (Figure 9c,d). Here no or only small amounts of adsorbed protein were removed by changing the pH. This is an additional hint toward the noteworthiness of the adsorption of R-chymotrypsin in the lowsalt buffer at pH 9. The experiments devoted to the investigation of the switchability and reversibility of protein adsorption will be continued and complemented by additional experimental regimes (offer of additional protein after switching the surrounding pH) and methods such as fluorescence microscopy and in-situ ATR (attenuated total reflection FTIR).

5. Conclusions P2VP-PAA mixed brushes (i.e., brushes of two oppositely charged polyelectrolytes) were shown to be a versatile tool to use in tuning the surface properties at biointerfaces. Depending (62) Fink, A. L. In Encyclopedia of Life Sciences; Nature Publishing Group: London, 2001. (63) Baszkin, A., Norde, W., Eds. Physical Chemistry of Biological Interfaces; Marcel Dekker: New York, 2000.

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Figure 9. Reversibility test of protein adsorption by changing the pH of the surrounding low-salt buffer: adsorption of R-chymotrypsin from a pH 9 buffer and subsequent change in the surrounding buffer to pH 4 (a); adsorption of R-chymotrypsin from pH 4 buffer and change to pH 9 buffer (b); adsorption of R-lactalbumin from pH 9 buffer and change to pH 4 buffer (c) and with an inverted pH regime (d).

on the pH and salt concentration of the surrounding aqueous solution, the interfacial properties exposed to biomolecules as proteins can be varied in terms of positive or negative charges, hydrophilicity/ hydrophobicity, and conformation of the polymer chains from stretched flexible chains to inflexible coil-like structures. The net charge of protein and brush, the confinement of counterions inside the brush, and the conformation of the polymer chains of the brush greatly influenced the protein adsorption of the two investigated globular model proteins. By changing the pH and/or the addition of salt, the amount and the mechanism of an adsorbing protein were completely changed. In other words, the brush regime also strongly influences protein adsorption: In the salted regime, protein adsorption was found to be similar to that often-described at hydrophobic surfaces. However, in the osmotic regime the balance of electrostatic

repulsion and a strong entropic driving force (i.e., counterion release) was found to be the main influence on protein adsorption. This driving force has, until now, been found only for colloidal homobrush surfaces and not at all for nonbrush polyelectrolyte interfaces. Acknowledgment. We thank Kathrin Po¨schel and Nicole Petong for their help during the performance of the ADSA experiments, Rene´ Winkler, Manuela Heber, and Thomas Schmidt for the preparation of the brushes and help with the ellipsometric experiments, Carsten Werner for allowing us to use the streaming ellipsometry setup in his department, and the DFG (SFB 287, B10, and STA324/17-2) for financial support. LA061557G