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pH-Dependent Immobilization of Proteins on Surfaces Functionalized by Plasma-Enhanced Chemical Vapor Deposition of Poly(acrylic acid)and Poly(ethylene oxide)-like Films Serena Belegrinou,† Ilaria Mannelli, Patricia Lisboa, Frederic Bretagnol, Andrea Valsesia, Giacomo Ceccone, Pascal Colpo, Hubert Rauscher,* and Franc¸ois Rossi Institute for Health and Consumer Protection, European Commission, Joint Research Centre, Via E. Fermi, 21027 Ispra (VA), Italy ReceiVed January 31, 2008. ReVised Manuscript ReceiVed March 30, 2008 The interaction of the proteins bovine serum albumin (BSA), lysozyme (Lys), lactoferrin (Lf), and fibronectin (Fn) with surfaces of protein-resistant poly(ethylene oxide) (PEO) and protein-adsorbing poly(acrylic acid) (PAA) fabricated by plasma-enhanced chemical vapor deposition has been studied with quartz crystal microbalance with dissipation monitoring (QCM-D). We focus on several parameters which are crucial for protein adsorption, i.e., the isoelectric point (pI) of the proteins, the pH of the solution, and the charge density of the sorbent surfaces, with the ζ-potential as a measure for the latter. The measurements reveal adsorption stages characterized by different segments in the plots of the dissipation vs frequency change. PEO remains protein-repellent for BSA, Lys, and Lf at pH 4-8.5, while weak adsorption of Fn was observed. On PAA, different stages of protein adsorption processes could be distinguished under most experimental conditions. BSA, Lys, Lf, and Fn generally exhibit a rapid initial adsorption phase on PAA, often followed by slower processes. The evaluation of the adsorption kinetics also reveals different adsorption stages, whereas the number of these stages does not always correspond to the structurally different phases as revealed by the D-f plots. The results presented here, together with information obtained in previous studies by other groups on the properties of these proteins and their interaction with surfaces, allow us to develop an adsorption scenario for each of these proteins, which takes into account electrostatic protein-surface and protein-protein interaction, but also the pH-dependent properties of the proteins, such as shape and exposure of specific domains.
Introduction The adsorption of proteins is important for numerous interdisciplinary scientific fields, such as biomaterial and biomedical science, biosensor development, or nanobiotechnology, because proteins tend to accumulate spontaneously at solid-liquid interfaces and attach to surfaces as tightly bound adsorbates.1,2 Irreversible and uncontrolled accumulation of proteins has a detrimental influence on a wide range of technological applications. For instance, biomedical devices and many implants in contact with biological media, in particular with blood, must consist of materials with low potential for protein adsorption and cellular interactions.3 Otherwise, there is the risk of adverse biological reactions in the living host system, such as acute and chronic inflammation or fibrous encapsulation. Medical devices which are in daily use, for instance contact lenses, are subject to fouling processes as well. Furthermore, bioadhesion is also relevant for nonbiomedical areas, e.g., systems for water purification, transport, and storage which acquire biofilms as well.4 On the other hand, protein adsorption can be desirable if it proceeds under controlled and well-characterized conditions. Well-controlled interactions between surfaces and biomolecules * To whom correspondence should be addressed. E-mail: hubert.rauscher@ jrc.it. † Present address: University of Basel, Department of Chemistry, Klingelbergstrasse 80, 4056 Basel, Switzerland. (1) Haynes, C. A.; Norde, W. Colloids Surf., B 1994, 2, 517. (2) Horbett, T. A, Proteins: structure, properties, and adsorption to surfaces. In Biomaterials science: An introduction to materials in medicine; Ratner, B. D., Hoffman, A. S., Schoen, F. J., Lemons, J. E. , Eds.; Academic Press: San Diego, 1996. (3) Lazos, D.; Franzka, S.; Ulbricht, M. Langmuir 2005, 21, 8774. (4) Kingshott, P.; Griesser, H. J. Curr. Opin. Colloid Interface Sci. 1999, 4, 403.
are crucial for the development and optimization of biomaterials, biosensors, and other bioanalytical in vivo applications. The interaction between a biological recognition element (e.g., proteins) and an inorganic transducer surface is one of the key aspects in biosensor technology, since it directly affects the sensitivity and specificity of biosensors.5 Many chromatographic separations are also based on differences in binding affinities of proteins to the support material.1 Therefore, a detailed understanding of protein adsorption on functionalized surfaces is necessary for the development and improvement of bioanalytical applications. However, protein-surface interactions are complex and not yet well understood due to the different forces interacting between the protein domains and the substrate, as well as the complex dynamic adsorption processes. Furthermore, there are multiple protein, solution, and surface variables which affect protein adsorption.6–9 In this study, we therefore focus on several parameters which are crucial for protein adsorption, i.e., the isoelectric point (pI) of the proteins, the molecular weight, the pH of the solution, and the charge density of the sorbent surfaces, with the ζ-potential as a measure for the latter. The selection of these parameters is driven by the goal of this study, namely to correlate the charge density of surfaces functionalized via plasmaenhanced chemical vapor deposition (PE-CVD) with the net charge of four different proteins between pH 4 and 8.5. (5) Marco, M.-P.; Barcelo´, D. Meas. Sci. Technol. 1996, 7, 1547. (6) Andrade, J. D. Surface and Interfacial Aspects of Biomedical Polymers, Vol. 2: Protein adsorption; Plenum Press: New York, 1985. (7) Andrade, J. D.; Hlady, V. L.; Van Wagenen, R. A. Pure Appl. Chem. 1984, 56, 1345. (8) Dee, K. C.; Puleo, D. A.; Bizios, R. An introduction to tissue-biomaterial interactions; Wiley-Liss: Hoboken, 2003. (9) Latour, R. A. Biomaterials: Protein-Surface Interactions. In Encyclopedia of Biomaterials and Biomedical Engineering; Bowlin, G. L., Wnek, G., Eds.; Taylor and Francis: New York, 2005; pp 1.
10.1021/la8003454 CCC: $40.75 2008 American Chemical Society Published on Web 06/13/2008
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Substrate Preparation. Polished AT-cut and SiO2-coated sensor quartz discs (14 mm in diameter, 0.30 mm thick) with a fundamental frequency of 5 MHz (Q-Sense AB, Gothenburg, Sweden) were used for the QCM-D experiments. The quartz crystals were cleaned by sequential immersion in 1 M NaOH, 1 M HCl, acetone, and ethanol for 10 min at a time. For the ζ-potential measurements, double-sided polished silicon wafers (N-type doping with P, (100) orientation, thickness 280 µm ( 25 µm; Institute of Electronic Materials Technology, Warsaw, Poland) and glass microslides (76 mm × 26 mm; Forlab, Brussels, Belgium) were used. The cleaning procedure consisted of sequential sonication (5 min per cycle) in trichloroethane, acetone, and ethanol.
Plasma Polymerization via PE-CVD. PEO-like and PAA coatings, both deposited via PE-CVD, were chosen as substrates that are resistant to protein adsorption (PEO) or that promote the adsorption of proteins (PAA). These coatings have been characterized earlier, including some aspects regarding their interaction with biomolecules.10–22 The PEO-like coatings used in this work were produced in a custom built, capacitively coupled plasma source within a stainlesssteel reactor. Two parallel plate electrodes were mounted in the interior with a distance of 5 cm. The bottom electrode was grounded and acted as a sample holder, while the upper electrode was connected to a radio frequency power source generator (13.56 MHz). The depositions were performed with an input power of 5 W in continuous mode for 25 s, and were then continued in pulsed mode with 10% duty cycle for 80 min, which led to films with a thickness of ∼25 nm as measured by ellipsometry. Diethylene glycol dimethyl ether (DEGDME; Sigma-Aldrich, purity g99.5%) was used as a poly(ethylene glycol)-like monomer precursor with a flow rate of 3 sccm (standard cubic centimeters), at a working pressure of 12 mTorr during the deposition. The pressure was monitored by a baratron (MKS). As DEGDME has a relatively high boiling point (162 °C), the connecting gas pipe between the monomer storage vessel and the reaction chamber was heated to avoid condensation. The cleaning procedure between each deposition consisted of 15 min of pure oxygen plasma, followed by 10 min of pure argon plasma, both applied in continuous mode. From earlier studies it is known that layers produced in such a way are resistant to protein adsorption.16 The PAA films for this study were produced in a home-built capacitively coupled plasma reactor (13.56 MHz excitation frequency). In contrast to the reactor described above, the bottom electrode, which served as sample holder, was connected to the power source. The upper, grounded electrode had a “showerhead” design to admit the gas mixture, which consisted of 5 sccm acrylic acid (Sigma-Aldrich, g99%) and 5 sccm argon. The depositions were carried out for 1 min at an input power of 50 W in pulsed mode with 10% duty cycle and 4 ms “time on” and led to films with a thickness of ∼30 nm. Between each deposition the chamber was plasma-cleaned with a mixture of 5 sccm argon and 5 sccm oxygen in continuous mode (24 W) for 15 min. PAA films produced this way promote protein adsorption.23 Both types of PE-CVD functionalized surfaces were characterized by X-ray photoelectron spectroscopy (XPS) after 12 h of immersion of the coated samples in 10 mM phosphate buffered saline (PBS) solution in the investigated pH range, as well as after immersion in deionized water. The photoelectron spectra did not indicate any change of the film by the immersion procedure. Furthermore, corresponding ellipsometry measurements before and after immersion showed that the layers are stable in solution, and swelling of the films could be excluded. These results indicate that changes in the dissipation and the film thickness as measured by QCM-D upon exposure to the protein solutions must be due to the adsorption of proteins and is not caused by changes of the PAA- or PEO-like films. ζ-Potential Measurements. The ζ-potential is defined as the potential which arises at the shear plane between a bulk electrolyte solution and a surface when there is a relative movement between the liquid and the solid phase.24 It is affected by the dissociation of
(10) Johnston, E. E.; Bryers, J. D.; Ratner, B. D. Langmuir 2005, 21, 870. (11) Valsesia, A.; Colpo, P.; Meziani, T.; Bretagnol, F.; Lejeune, M.; Rossi, F.; Bouma, A.; Garcia-Parajo, M. AdV. Funct. Mat. 2006, 16, 1242. (12) Candan, S.; Beck, A. J.; O’Toole, L.; Short, R. D. J. Vac. Sci. Technol. A 1998, 16, 1702. (13) Sardella, E.; Gristina, R.; Senesi, G. S.; d’Agostino, R.; Favia, P. Plasma Process. Polym. 2004, 1, 63. (14) Sardella, E.; Gristina, R.; Ceccone, G.; Gilliland, D.; PapadopoulouBouraoui, A.; Rossi, F.; Senesi, G. S.; Detomaso, L.; Favia, P.; d’Agostino, R. Surf. Coat. Technol. 2005, 200, 51. (15) Sardella, E.; Favia, P.; Gristina, R.; Nardulli, M.; d’Agostino, R. Plasma Process. Polym. 2006, 3, 456. (16) Bre´tagnol, F.; Lejeune, M.; Papadopoulou-Bouraoui, A.; Hasiwa, M.; Rauscher, H.; Ceccone, G.; Colpo, P.; Rossi, F. Acta Biomater. 2006, 2, 165. (17) Bre´tagnol, F.; Kylia´n, O.; Hasiwa, M.; Ceriotti, L.; Rauscher, H.; Ceccone, G.; Gilliland, D.; Colpo, P.; Rossi, F. Sens. Actuat. B 2007, 123, 283.
(18) Bre´tagnol, F.; Ceriotti, L.; Lejeune, M.; Papadopoulou-Bouraoui, A.; Hasiwa, M.; Gilliland, D.; Ceccone, G.; Colpo, P.; Rossi, F. Plasma Process. Polym. 2006, 3, 30. (19) Rossini, P.; Colpo, P.; Ceccone, G.; Jandt, K. D.; Rossi, F. Mater. Sci. Eng., C 2003, 23, 353. (20) Valsesia, A.; Manso Silvan, M.; Ceccone, G.; Gilliland, D.; Colpo, P.; Rossi, F. Plasma Process. Polym. 2005, 2, 334. (21) Favia, P.; Sardella, E.; Gristina, R.; d’Agostino, R. Surf. Coat. Technol. 2003, 707, 169–170. (22) Muir, B. W.; Tarasowa, A.; Gengenbach, T. R.; Menzies, D. J.; Meagher, L.; Rovere, F.; Fairbrother, A.; McLean, K. M.; Hartley, P. G. Langmuir 2008, DOI: 10.1021/la702689t. (23) Lejeune, M.; Valesia, A.; Kormunda, M.; Colpo, P.; Rossi, F. Surf. Sci. 2005, 583, L142. (24) Hunter, R. J. Foundations of colloid science, 2nd ed.; Oxford University Press: New York, 2005.
The PE-CVD method was chosen for surface modification, as it offers the possibility to obtain different tightly adherent films on a wide variety of substrates.10 The deposited films are, on the one hand, a protein resistant, poly(ethylene oxide)-like (PEOlike) coating and, on the other hand, a protein adsorbing, poly(acrylic acid) (PAA) surface. Earlier ζ-potential measurements on such surfaces revealed that these two surface types carry a negative charge at neutral pH.11 A property which is particularly important for this study is the pI of proteins, i.e., the pH at which the protein exhibits net zero charge. At a pH below the pI, proteins are positively charged, while they carry a negative net charge above it. Hence, if the protein-surface interaction is dominated by electrostatic forces, adsorption on a negatively charged surface should be more favorable at a pH below the pI’s of the proteins. In addition to ionic or electrostatic forces, the adsorption of proteins is affected by other interaction mechanisms, such as hydrophobic interactions or hydrogen bonding. To understand the interaction of biomolecules with functionalized surfaces in detail, it is desirable to realize whether one of the mechanisms is dominating the protein surface interaction in a certain pH range. An objective of this study is therefore to find out whether there is a direct correlation between the surface charge, the molecule charge, and the pH of the solution or whether other mechanisms dominate the adsorption process under specific conditions. To tackle these questions, we have chosen four different proteins and investigated their pH dependent adsorption behavior by quartz crystal microbalance with dissipation monitoring (QCM-D). The proteins are bovine serum albumin (BSA), lysozyme (Lys), lactoferrin (Lf), and fibronectin (Fn), which differ in size, molecular weight, and pI. The measurements reveal different adsorption stages and allow conclusions on the adsorption kinetics and on structural changes which are probably ongoing during the formation of the adsorbate layer. In conjunction with the ζ-potential results, these measurements provide information relevant to the understanding of the interaction between biomolecules and bioactive surfaces.
Materials and Methods
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Table 1. Molecular Weights and pI’s of the Proteins Used for This Study Mw [kDa] pI
BSA
Lys
Lf
Fn
66.0 4.5
14.7 11
90.0 8.7
450 5.0
surface groups or the adsorption of ions.25 Since there is no direct way to directly determine the surface potential or the surface charge density, the ζ-potential, which is accessible to measurements, can be used as a measure for both of these quantities.26 It can in turn be used to gain information on the dissociation of surface groups or the adsorption of cations or anions.25 Here we use the streaming potential to measure the ζ-potential by applying a flow of liquid under a pressure gradient along the sample. The potential difference generated along the sample is measured and used to determine the ζ-potential of the surface from the Smoluchowski equation27 ζ ) (dUSr/dp)(η/εε0)(L/QR), where USr is the streaming potential, p the pressure, η the viscosity, ε the dielectric constant, L and Q the capillary length and cross-section area, and R the electric resistance. By using a very diluted electrolyte solution, we can apply the Fairbrother-Mastin28approach, where the term L/QR is substituted by the conductivity of the electrolyte solution κ )L/(QR). Before measuring the ζ-potential, the plasma polymer coated samples were washed by immersion in deionized water for 12 h and then dried under nitrogen flow. The measurements were made with an electrokinetic analyzer (Anton Paar, Graz, Austria), using a clamping cell for solid samples connected to the instrument. Inside the cell, the sample was pressed against a PMMA spacer with seven rectangular channels. Therefore, the measured ζ-potential includes a contribution from the PMAA spacer, which can, however, be eliminated by measuring a reference PMAA surface. For this purpose, a PMMA reference curve was determined by measuring the ζ-potential of a PMMA reference plate under the same measuring conditions, under which the coated samples were investigated. Starting at pH 9, the raw ζ-potential values for each sample, as well as for the PMMA reference curves, were read in steps of 0.5 pH units. From these measurements, the ζ-potential values, ζs, of the sample surfaces were determined by using the equation ζs ) 2ζm - ζPMMA, where ζm and ζPMMA are the raw values measured against the PMMA reference and the ζ-potential values of the PMMA reference plate itself, respectively. The measurements were performed with 450 mL of a 1 mM KCl solution. The pH was initially adjusted to ∼10 by adding 0.1 M NaOH solution. During the measurement, the pH was lowered stepwise (∼0.5 pH units) by automatic titration with a 0.1 M HCl solution until pH ∼2 was reached. During the measurement, a pressure ramp in the range of 0-600 mbar was applied. In order to ensure good statistics, the ζ-potential value at a certain pH was the average of four single measurement points. Protein Samples. The proteins used in this study are listed in Table 1, together with those features which are most important for this study, i.e., molecular weight (Mw) and pI.29–31 The proteins are bovine serum albumin, (BSA, Sigma Aldrich, A7906), lysozyme from chicken egg white (Lys, Fluka, 62971), lactoferrin, isolated from bovine colostrum (Lf, Sigma Aldrich, L4765), and fibronectin from bovine plasma (Fn, Sigma Aldrich, F4759). The proteins were used without further purification. PBS solutions (10 mM) at different pH were used for preparing protein solutions with concentrations of 50 µg/mL for interaction with PAA coatings and 100 µg/mL for the PEO coatings. The pH of the PBS solution was preadjusted by (25) Jacobasch, H.-J. Progr. Org. Coat. 1989, 17, 115. (26) Bellmann, C.; Klinger, C.; Opfermann, A.; Bo¨hme, F.; Adler, H.-J. P. Prog. Org. Coat. 2002, 44, 93. (27) Jacobasch, H.-J.; Simon, F.; Werner, C.; Bellmann, C. Technisches Messen 1996, 63, 439. (28) Fairbrother, F.; Mastin, H. J. Chem. Soc. Trans. 1924, 125, 2319. (29) Wadu-Mesthridge, K.; Amro, N. A.; Liu, G.-Y. Scanning 2000, 22, 380. (30) Levay, P. F.; Viljoen, M. Haematologica 1995, 80, 252. (31) McArthur, S. L.; McLean, K. M.; Kingshott, P.; St John, H. A. W.; Chatelier, R. C.; Griesser, H. J. Colloids Surf., B 2000, 17, 37.
adding aqueous NaOH or HCl solution until the desired pH values of approximately 4, 5.5, and 8.5 were reached. For the measurements at pH ∼7, pure, unadjusted PBS (pH ∼7.3) was used. Quartz Crystal Microbalance with Dissipation Monitoring. The interaction between proteins and PE-CVD functionalized surfaces was investigated utilizing the QCM-D technique, by monitoring both frequency and dissipation changes simultaneously in real time during adsorption. Rigidly bound adlayers can precisely follow the crystal’s oscillatory motion, while weakly bound layers cannot and consequently induce dissipative losses. In this way it is possible to distinguish individual adsorption stages and to detect structural changes, such as spreading or cluster formation of the adsorbed protein films.32–34 The instrument was a QCM-D E4 (Q-Sense AB, Gothenburg, Sweden), which uses four temperature-stabilized measurement cells (in parallel configuration in our case). The QCM instrument was able to record frequencies up to the 13th overtone. All graphs presented in this study show the fifth overtone, which is little sensitive to variations of the mounting conditions of different crystals. After coating the quartz crystals by PE-CVD, they were immersed in deionized water for 12 h to remove the surplus of monomer and physisorbed material. They were then assembled into the QCM and the functionalized surfaces were first exposed to 10 mM PBS solution in order to stabilize the system for at least 20-30 min. Once stable frequency and dissipation signals were recorded, the protein solution was pumped through the measurement chamber by applying a flow of 50 µL/min. The frequency and dissipation changes due to adsorption of proteins were monitored until stable signals were recorded at saturation. If strict saturation was unattainable, the measurement was continued until the frequency and the dissipation curves did not shift by more than 1-2 Hz per 30 min. The last step of the experiments was a rinsing step with PBS solution to remove weakly bound or physisorbed material from the surface. The frequency differences obtained by subtracting the frequency value recorded just before the injection of the protein solution from the one after the final PBS rinsing were used for further evaluation, e.g., conversion of the frequency shift (∆f) into mass (∆m) adsorbed on the active electrode surface area (∼0.2 cm2). All QCM-D adsorption experiments were performed at 25 ( 0.02 °C and repeated at least twice. The reagents used for preparing working or buffer solutions were KCl and NaOH from Fluka and HCl and PBS from SigmaAldrich.
Results and Discussion ζ-Potential Measurements. In order to ascertain the reproducibility of the ζ-potential results, both the PEO-like and the PAA coatings were measured at least six times. As electrokinetic measurements are highly sensitive toward changes in the environmental conditions, the measurement conditions have to be controlled carefully.35 Therefore, for one set of samples, the plasma polymerized coating was deposited in the same run and electrolyte solution from the same batch was used. This resulted in a very good reproducibility regarding the shape of the curve, the location of the point of zero charge (PZC) and the ζ-potential values. For instance, at any measured pH the maximum deviation of the ζ-potential values of the PEO-like coatings was ∼3 mV and the PZCs diverge at most by 0.5 units in different measurements. For the PAA samples, maximum differences of 8 mV were obtained for the ζ-potential values and the PZCs had the same uncertainty as in the measurements with the PEO-like (32) Keller, C. A.; Glasma¨star, K.; Zhdanov, V. P.; Kasemo, B. Phys. ReV. Lett. 2000, 84, 5443. (33) Reimhult, E.; Ho¨o¨k, F.; Kasemo, B. Langmuir 2003, 19, 1681. (34) Rodahl, M.; Ho¨o¨k, F.; Fredriksson, C.; Keller, C. A.; Krozer, A.; Brzezinski, P.; Voinova, M. V.; Kasemo, B. Faraday Discuss. 1997, 107, 229. (35) Hinze, F.; Ripperger, S.; Stintz, M. Chem. Ing. Technik. 1999, 71, 338. (36) McFadyen, P. Int. Labmate 2002, 27, 26. (37) Temmel, S.; Kern, W.; Luxbacher, T. Prog. Colloid Polym. Sci. 2005, 132, 83.
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Figure 1. ζ-potential results for PEO-like and PAA surfaces prepared by PE-CVD.
coatings (0.5 units). The absolute magnitude of the measured ζ-potentials varied slightly for a series of samples, but such differences are usually of little consequence.36 Figure 1 shows typical results of the ζ-potential measurements for the PEO-like films and for the PAA films, respectively, already corrected for the contribution of the PMAA spacer. The PZC of the PEO-like surface is located at pH 3.7, which is typical for polymeric surfaces without dissociable functional groups.37 The negative surface charge above the PZC arises from preferential adsorption of chloride and hydroxyl anions. The PAA coating has the PZC at pH 2.7, one unit below the one of the PEO film. For PAA, the ζ-potential depends more strongly on the pH than for PEO since dissociation of the carboxylic functional groups contributes to the formation of the electrochemical double layer (EDL), in addition to the adsorption of anions. As the PZC can also serve as a measure of acidity and alkalinity of solid surfaces,38 the low PZC value of the PAA coating confirms its stronger acidity compared to the PEO surfaces. The dissociation of the carboxylic groups also affects the shape of the ζ-potential curves and the values at large pH. A clear plateau in the alkaline range (pH 8-9), which is the result of complete dissociation of carboxylic functional surface groups, can be observed for the PAA sample. The ζ-potential values in this range reach -85 mV and are much more negative than those for PEO, which do not exceed -30 mV. These results show that in the pH region between 4 and 8.5, in which the protein adsorption experiments were performed, both surfaces carry a negative surfaces charge while the PAA films are more negative than the PEO-like films. Hence, if electrostatic interactions are the main driving forces for protein adsorption on the investigated surfaces, proteins are expected to adsorb readily on PAA at a working pH below their pI’s. QCM-D Measurements. BSA Adsorption on PAA. The frequency, ∆f (Figure 2a), and the dissipation, ∆D (Figure 2b), shifts of a PAA-coated QCM sensor upon exposure to BSA are shown in figure 2 as a function of time. The arrows indicate the start of the exposure to the BSA solution and later the start of the rinsing step with PBS. The rinsing confirms that the adsorption of BSA is irreversible under these experimental conditions for all investigated pH values, since rinsing induces almost no changes of the frequency or of the dissipation. A summary of the frequency and dissipation shifts is presented in table 2. From these values, the adsorbed mass, ∆m, and the
resulting thickness of the films, dSauerbrey, were calculated via the Sauerbrey equation ∆m ) -C∆f/n, where C ) 17.7 ng/cm2 · Hz at f ) 5 MHz, and n is the overtone number (here n ) 5). Alternatively, the film thickness, dfit, was also calculated by applying a viscoelastic, single-layer Voight model to the ∆f and ∆D response as implemented in the QCM software and described in detail by Voinova et al.39 Results obtained from both models and the number of adsorbed molecules, nBSA, per unit area calculated from these results are also included in Table 2. Generally, the values obtained from fitting with the Voight model and those from the Sauerbrey equation are in good agreement, with the exception of those for pH 5.5. At pH 4, the adsorption is fast and saturation is achieved rapidly (Figure 2a). The relatively small dissipation value obtained at saturation (Figure 2b) indicates that the adlayer is rigidly bound under these conditions. At pH above 4 the adsorption rates are smaller and saturation requires more time with increasing pH
(38) Bismarck, A.; Kumru, M. E.; Springer, J. J. Colloid Interface Sci. 1999, 217, 377.
(39) Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B. Phys. Scr. 1999, 59, 391.
Figure 2. ∆f (a) and ∆D (b) vs time plot for the adsorption of BSA on PAA. Black, data for pH 4; red, pH 5.5; blue, pH 7; green, pH 8.5. The arrows in (a) indicate the start of the exposure to the BSA solution (∆f ) 0 Hz) and the start of the rinsing step with PBS. (c) ∆D vs ∆f plot obtained from these data.
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Table 2. Compilation of Data Obtained for the Interaction of BSA with PAA (see Figure 2a-c) and Fitted Dataa ∆f [Hz] ∆D × 10-6 ∂D/∂ft ) 1 × 10-9 [Hz-1] ∂D/∂ft ) 2 × 10-9 [Hz-1] D/∂ft ) 3 × 10-9 [Hz-1] k1 [s-1] k2 [s-1] k3 [s-1] dSauerbrey [nm] dfit [nm] ∆m [ng/cm2] nBSA × 1012
pH 4
pH 5.5
pH 7
pH 8.5
24.7 0.35 8.7 28.5 1.4 × 10-2 ( 2.7 × 10-4 1.0 × 10-3 ( 1.3 × 10-4 4.3 4.4 429 3.9
38.6 0.92 27.1 15.9 2.7 × 10-3 ( 4.9 × 10-5 7.5 × 10-4 ( 3.9 × 10-5 6.8 10.3 677 6.2
24.8 1.17 79.8 43.5 16.5 4.2 × 10-3 ( 2.8 × 10-4 1.3 × 10-3 ( 3.9 × 10-5 3.5 × 10-4 ( 1.0 × 10-4 4.4 4.5 439 4.0
11.6 0.80 80.5 51.0 7.9 × 10-4 ( 3.8 × 10-6 9.8 × 10-5 ( 1.2 × 10-6 2.1 2.3 208 1.9
a The indices t ) 1-3 of the slopes ∂D/∂f refer to possible different adsorption stages apparent in Figure 2c, while the indices of the kinetic constants, k, refer to kinetically different adsorption processes used for the fitting.
value. The adsorption maximum for BSA is reached at pH 5.5, while adsorption at pH 8.5 yields the smallest frequency shift (see Table 2 and Figure 2a). At pH 7 the frequency shift at saturation is almost the same as at pH 4, but the adsorption is slower and the dissipation at pH 7 is more than 3 times larger than for pH 4.
Figure 3. ∆f (a) and ∆D (b) vs time plot for the adsorption of lysozyme on PAA. Black, data for pH 4; red, pH 5.5; blue, pH 7; green, pH 8.5. (c) ∆D vs ∆f plot obtained from these data.
These findings are in agreement with previous studies on pHdependent adsorption of BSA on other surfaces which showed maximum adsorption close to the pI.40–42 At its pI, and particularly on a hydrophilic surface43 such as PAA, BSA will undergo very little conformational change40 which allows adsorption in its normal, compact conformation, leading to a compact layer. At pH >4.5, i.e., below pI, the conformation (R-helix content) changes more pronouncedly than at larger pH,40 and therefore, the amount of adsorbed BSA decreases sharply below pI due to (i) the expanded conformation which BSA adopts at low pH and (ii) stronger intermolecular repulsion. The expanded conformation of BSA at pH >4.5 prevents the adsorption of a larger amount of BSA due to steric hindrance. In contrast, the loss of R-helix content is moderate at higher pH,44 which allows the same amount of BSA adsorption at pH 7 and 4. As discussed above, under the adsorption conditions employed here, the ζ-potential of the PAA surface is negative, which indicates that the surface carries a net negative charge. However, since the pI of BSA is around 4.5, the molecules carry only little net positive charge at pH 4 which should not lead to strong electrostatic interaction with PAA. At a pH close to the pI, on the other hand, proteins generally exhibit a high surface activity8 because under these conditions electrostatic repulsive interactions are significantly reduced in favor of hydrophobic attractive interactions. Considering also that BSA is close to its normal, compact shape at pH not too much above the pI, with only little loss of R-helix content, it is not surprising that the largest amount of BSA is accumulated on the surface at pH 5.5. It appears therefore that from pH 4 up to at least pH 7 electrostatic interaction between BSA and the surface plays a minor role. Notwithstanding the fact that pH 8.5 is relatively far from the pI of BSA, the protein still has its normal configuration under these conditions,44 and therefore, the formation of a compact layer should not be hindered by steric reasons at this pH. The decrease of the adsorbable amount of BSA at higher pH must therefore have other reasons. At that pH, the protein and the PAA film carry a negative net charge. Hence, repulsive electrostatic interactions are expected between the molecules and the surface and also between the individual molecules. This leads to less adsorption as the pH increases, although it is obvious that adsorption still proceeds at pH 8.5. Therefore, at high pH electrostatic interaction becomes more dominating, but other (40) Peng, Z. G.; Hidajat, K.; Uddin, M. S. Colloids Surf., B Biointerfaces 2004, 33, 15. (41) Hu, J.; Li, S.; Liu, B. Biochem. Eng. J. 2005, 23, 259. (42) Giacomelli, C. E.; Avena, M. J.; De Pauli, C. P. J. Colloid Interface Sci. 1997, 188, 387. (43) Wertz, C. F.; Santore, M. M. Langmuir 2001, 17, 3006. (44) Foster, J. F. In Albumin Structure, Function and Uses, Rosenoer, V. M., Oratz, M., Rothschild, M. A.,Eds. Pergamon: Oxford, 1977; pp 53.
7256 Langmuir, Vol. 24, No. 14, 2008
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Table 3. Summary of the ∆f and ∆D Values for Lysozyme Adsorption on PAAa ∆f[Hz] ∆D × 10-6 ∂D/∂f × 10-9 [Hz-1] k1 [s-1] k2 [s-1] dSauerbrey [nm] dfit [nm] ∆m [ng/cm2] nLys × 1012 a
pH 4
pH 5.5
pH 7
pH 8.5
8.8 0.04 277.1 5.0 × 10-2 ( 1.1 × 10-3 1.6 1.5 160 6.6
11.7 0.05 57.4 4.6 × 10-2 ( 1.5 × 10-3 1.9 2.0 207 8.5
15.3 0.04 18.7 1.1 × 10-2 ( 1.3 × 10-4 2.9 × 10-4 ( 4.9 × 10-6 2.8 2.5 273 11.2
12.2 0.05 14.8 8.8 × 10-3 ( 8.6 × 10-5 3.0 × 10-4 ( 2.2 × 10-6 2.1 2.0 212 8.7
The slopes of the D-f plot are also included, as well as the results of the different model calculations.
types of attractive interactions, e.g. hydrophobic, may still enable protein adsorption at high pH.45 In order to gain more information on the adsorption behavior, ∆D is plotted against ∆f in a D-f plot. This way of plotting eliminates the time as an explicit parameter and can be used to obtain additional information on the adsorption processes via an analysis of the slopes ∂D/∂f.46–49 The D-f plots for BSA adsorption on PAA are shown in Figure 2c. All ∆D vs ∆f curves, except the one for pH 8.5, show segments with different, approximately constant slopes, highlighted by the colored arrows, which indicates that there are different adsorption phases. In the initial phase, the D-f plot for pH 4 is linear with a small slope. The distant data points indicate a fast initial adsorption phase. The initial phase is terminated by steric restrictions of adsorbed BSA which tends to spread at low pH to adopt a more expanded form.40,43 The slope ∂D/∂ft ) 2 for the second adsorption phase is 3.3 times larger than in the initial phase, which points toward the formation of an adlayer with higher dissipation per added mass. It indicates that the adsorbed BSA molecules are mobile enough to allow insertion of additional molecules into the layer after the first, rapid adsorption step and that reorientation processes, as suggested earlier,43 may also take place. In contrast to pH 4, the slopes of the D-f plots for pH 5.5 and 7 decrease as the adsorption proceeds because the dissipation per added mass is small near saturation. In the case of pH 5.5, the slope in the final adsorption phase is close to zero, i.e., at this stage the addition of mass does not change the dissipation anymore. Obviously, under these conditions a very rigid protein layer is formed near saturation. This finding is in agreement with the view that BSA, at least at pH 5.5 and 7, i.e., not too much above its pI, adsorbs in its native, compact conformation which leads to a compact layer. Finally, at pH 8.5 it is not possible to clearly distinguish different adsorption stages because the D-f plot has no clear break in the slope, although the latter slightly changes as the adsorption proceeds. The kinetics of the different adsorption stages as indicated by the D-f plots also were also analyzed by modeling the adsorption curves using different simple kinetic models. In the calculations, we assumed that the protein uptake is adsorption limited and irreversible, i.e., desorption processes were neglected. For instance, the initial part of the adsorption curve at pH 8.5 could be fitted very well with an exponential model of the form ∆f ) C1 exp(-tk1) + f0 with k1 ) 7.9 × 10-4 s-1, which corresponds to the finding from the D-f plot that there is only one adsorption process at pH 8.5. On the other hand, and taking into account also later adsorption stages, it is also possible to model the data (45) Belfort, G.; Lee, C. S. PNAS 1991, 88, 9146. (46) Fredriksson, C.; Kihlman, S.; Rodahl, M.; Kasemo, B. Langmuir 1998, 14, 248. (47) Ho¨o¨k, F.; Rodahl, M.; Brzezinski, P.; Kasemo, B. Langmuir 1998, 14, 729. (48) Ho¨o¨k, F.; Rodahl, M.; Kasemo, B.; Brzezinski, P. PNAS 1998, 95, 12271. (49) Oetzen, D. E.; Oliveberg, M.; Ho¨o¨k, F. Colloids Surf., B 2003, 29, 67.
with a sum of two exponential functions of the form ∆f ) C1 exp(-tk1) + C2 exp(-tk2) + f0, with a slightly modified k1 (k1 ) 7.5 × 10-4 s-1) and k2 ) 9.8 × 10-5 s-1, respectively. The latter constant points toward a very slow, additional adsorption process. However, at this late stage of film formation, the desorption of molecules may not be negligible anymore. In this case only the fit to the initial part of the adsorption curve would represent the actual adsorption kinetics. The adsorption curves for pH 4 and 5.5 could be fitted with two exponential functions each, corresponding to the two adsorption stages which are indicated by the two segments of constant slope in the D-f plot. For pH 7, the best fit could be achieved by applying a model comprising three exponential functions, in agreement with the three segments in the D-f plot for pH 7. The results of the kinetic modeling are summarized in Table 2. The largest kinetic constant k1 for the adsorption of BSA on PAA of 1.4 × 10-2 s-1 was obtained for the initial adsorption phase at pH 4. Lysozyme Adsorption on PAA. The frequency and dissipation curves of the lightest protein, lysozyme, are presented in Figure 3a and b. The adsorption of lysozyme is characterized by an initial rapid frequency decrease for all investigated pH values. For pH 4, the frequency remains nearly constant after the first initial decrease, whereas for pH 5.5, 7, and 8.5 there are subsequent slower adsorption phases. Hence, under these conditions, saturation is reached only at pH 4. Unlike BSA, lysozyme adsorbs partly reversibly at all pH values. During the washing step, the layers deposited at the lower pH values 4 and 5.5 have the highest losses (corresponding to an increase of ∼5 Hz in frequency) whereas the frequency of the curves at pH 7 and 8.5 increases only by 3 and 2 Hz, respectively. As the investigated range of pH is below the pI of lysozyme, electrostatic attraction between the surface and the molecules is assumed to be relevant for all investigated pH values, particularly on a hydrophilic surface,50 such as PAA. This view is in agreement with the earlier finding that adsorption of this protein is facilitated by predominantly electrostatic interaction between lysozyme and a surface covered by a -COOH-terminated self-assembled monolayer.29 Our experimental results show that in the initial phase, the adsorption is very fast at pH 4 and 5.5 and the initial frequency change is larger than at higher pH, which is in agreement with the view that electrostatic interactions are dominating, as the protein is strongly positively charged at these pH values. In addition to a strong electrostatic interaction, rapid adsorption of the globular protein lysozyme is facilitated by its low electric dipole moment (∼300 D at pH 5 in water51), which allows the
1.
(50) Lu, J. R.; Su, T. J.; Howlin, B. J. J. Phys. Chem. B 1999, 103, 5903. (51) Bonincontro, A.; De Francesco, A.; Onori, G. Colloids Surf., B 1998, 12,
pH-Dependent Immobilization of Proteins
Langmuir, Vol. 24, No. 14, 2008 7257
Table 4. Results Obtained for Adsorption of Lactoferrin on PAA ∆f [Hz] ∆D × 10-6 ∂D/∂ft ) 1 × 10-9 [Hz-1] ∂D/∂ft ) 2 × 10-9 [Hz-1] ∂D/∂ft ) 3 × 10-9 [Hz-1] k1 [s-1] k2 [s-1] dSauerbrey [nm] dfit [nm] ∆m [ng/cm2] nLf × 1012
pH 4
pH 5.5
pH 7
pH 8.5
33.5 0.43 23.4 7.1 4.0 × 10-2 ( 6.5 × 10-4 5.9 7.3 589 3.9
47.9 0.38 29.0 -18.8 39.7 1.8 × 10-2 ( 1.3 × 10-4 6.0 × 10-4 ( 2.0 × 10-5 8.5 8.9 849 5.7
50.9 0.27 32.3 -20.6 22.8 1.3 × 10-2 ( 2.1 × 10-4 3.1 × 10-4 ( 6.2 × 10-5 9.0 8.5 901 6.0
47.4 0.50 31.5 -18.3 42.5 1.5 × 10-2 ( 1.9 × 10-4 3.4 × 10-4 ( 4.6 × 10-4 8.4 8.0 841 5.6
molecule to approach the PAA surface in random orientation52 without the need for reorientation due to the interaction with the charged surface. A rapid initial decrease has also been observed earlier for lysozyme adsorption on a hydrophilic TiO2 surface.53 The higher pH values 7 and 8.5 also exhibit a fast initial adsorption phase with subsequent slower phases. As a trend for lysozyme the slower adsorption phase starts earlier when the pH is higher. Concerning hydrophobic protein-surface interaction it was shown that exposed, freely accessible amino acids, such as Trp62, can interact with apolar surfaces through hydrophobic interactions.52 Interaction with a hydrophobic surface causes perturbation of the molecule and leads to slow structural changes in the secondary structure of the molecule.52,54 On the other hand it was found that adhesive forces due to hydrophobic interaction between lysozyme and wettable (hydrophilic) surfaces are low.55 Hence, they are not expected to dominate the interaction between lysozyme and PAA under our conditions. In addition, it has been found that Lys, as a small globular protein, has a low compressibility and tends to retain a robust, compact conformation on the surface.55 This taken together confirms the view that the interaction between lysozyme and the PAA surface under our experimental conditions is predominantly electrostatic. Lysozyme, with a relatively high pI (∼11), carries a net positive charge in the entire investigated range of pH. Therefore, electrostatic intermolecular repulsion8 weakens the adsorption and tends to destabilize the adsorbate layer, which is probably the reason for the loss of adsorbed mass during the rinsing step. This is supported by the fact that the smallest amount of adsorbed mass is lost during the washing step at pH 8.5, which is closest to the pI of lysozyme. At pH 4 and 5.5 lysozyme should be more positively charged than at pH 7 and 8.5, and therefore, the loss of adsorbed lysozyme is larger at lower pH values. Further, after rinsing the smallest frequency shift is obtained at pH 4. As both adsorbed and free lysozyme molecules exhibit a strong positive net charge at pH 4, the surface may not be covered completely anymore by the protein after the rinsing step. In addition, it was suggested that far from the pI, at pH
