2054
Langmuir 2004, 20, 2054-2056
A New Interpretation of Serum Albumin Surface Passivation Beata Sweryda-Krawiec, Halagowder Devaraj, George Jacob, and James J. Hickman* Department of Bioengineering, Clemson University, Clemson, South Carolina 29634-0905 Received May 20, 2003. In Final Form: September 6, 2003 We report our efforts to understand the passivation behavior of bovine serum albumin (BSA) and its structural changes at interfaces using X-ray photoelectron spectroscopy and contact angle measurements. The work investigated protein adsorption on two surfaces with widely different surface free energies. We investigated a hydrophilic surface represented by clean glass and a hydrophobic surface prepared by modifying a glass surface with a fluorinated self-assembled monolayer. The experiments indicate that, on the hydrophilic surface, BSA adsorbs in a two-step process and passivation of the surface is reached. Adsorption of BSA on the hydrophobic surface also continues until passivation of the surface. In contrast, however, the process occurs in a single step. Contact angle measurements show that at the completion of the adsorption process in both cases the same contact angle is reached despite the different adsorption behavior. We believe that this strongly indicates that the same outer surface composition is obtained for both surfaces despite different passivation routes. We postulate that there is a controlled loss of tertiary structure of BSA in descrete units that allows a specific structure to be formed on the surface that inhibits further protein adsorption regardless of the initial surface composition.
We wish to report on our efforts to understand the passivation behavior of bovine serum albumin (BSA) and its structural changes at interfaces using X-ray photoelectron spectroscopy (XPS) and contact angle measurements. The particular aim of this work was to investigate protein adsorption on two surfaces with widely different surface free energies to better understand which physical properties are important in describing the mechanism of serum albumin surface passivation. We investigated a hydrophilic surface represented by clean glass and a hydrophobic surface prepared by modifying a glass surface with a fluorinated self-assembled monolayer. We demonstrate here that, on the hydrophilic surface, BSA adsorbs in a two-step process and passivation of the surface is reached (i.e., no further adsorption is observed). Adsorption of BSA on the hydrophobic surface also continues until passivation of the surface. In contrast, however, the process occurs in a single step. Contact angle measurements show that, at the completion of the adsorption process, in both cases, the same contact angle is reached. We believe that this strongly indicates that the same outer surface composition is obtained for both surfaces despite different passivation routes. The structural changes of proteins at solid-liquid interfaces are of intense interest, especially for the serum albumins because of their unique surface passivation properties.1 However, the extent and rate of protein conformational changes on surfaces can be very difficult to measure.2,3 Some of the approaches attempted to date include spectral differences in proteins induced by adsorption4,5 and surface force techniques to provide information on the adsorbed layer thickness, softness of the * Author to whom correspondance should be addressed. (1) Nakanishi, K.; Sakiyama, T.; Imamura, K. J. Biosci. Bioeng. 2001, 91, 233. (2) Kodo, A.; Oko, S.; Higashitani, K. J. Colloid Interface Sci. 1991, 143 (1), 214. (3) Norde, W.; Farier, J. P. Colloids Surf. 1992, 64, 87. (4) Norde, W.; Giacomelli, C. E. J. Biotechnol. 2000, 779, 259. (5) Giacomelli, C. E.; Bremer, M. G.; Norde, W. J. Colloid Interface Sci. 1999, 220, 13.
adsorbed layer, and forces between adsorbed layers.6 Additional information on the advances in the field can be found in several review papers.7,8 XPS is an excellent surface-specific technique that can be used to study adsorbed protein layers on different surfaces because of its high surface sensitivity and chemical selectivity,9 and it has been utilized to study proteins deposited during cell culture.10 XPS has also been used in conjunction with time-of-flight secondary-ion mass spectrometry and other techniques for qualitative and quantitative investigations of protein adsorption on different surfaces.11 Contact angle measurements are a standard technique for determining the wetting properties of surfaces, which are directly proportional to the surface free energy.12 Micro-cover glasses (22 × 22 mm, no. 1, Thomas Scientific) were cleaned according to published procedures.13 The hydrophobic surface was prepared by modifying clean glass with trichloro(1H,1H,2H,2H-perfluorooctyl)silane (United Chemical Technology, Inc.).13 To ensure the desired surface properties, contact angle and XPS measurements were conducted, only samples with (6) (a) Wadu-Mesthrige, K.; Amro, N. A.; Liu, G. Scanning 2000, 22, 380. (b) Kawakami, H.; Takahashi, T.; Nagaoka, S.; Nakayama, Y. Polym. Adv. Technol. 2001, 12, 244. (c) Sheller, N. B.; Petrash, S.; Foster, M. D.; Tsukruk, U. V. Langmuir 1998, 14, 4535. (7) Nakanishi, K.; Sakiyama, T.; Imamura, K. J. Biosci. Bioeng. 2001, 91 (3), 233. (8) Fang, F.; Szleifer, I. Biophys. J. 2001, 80, 2568. (9) Slomkowski, S.; Kowalczyk, D.; Chehimi, M. M.; Dealamar, M. Colloid Polym. Sci. 2000, 278, 878. (10) Schaffner, A.; Barker, J.; Stenger, D.; Hickman, J. J. Neurosci. Methods 1995, 62, 111-119. (11) (a) Fitzpatrick, H.; Luckham, P. F.; Eriksen, S.; Hammond, K. J. Colloid Interface Sci. 1992, 149 (1), 1. (b) Lhoest, J. B.; Detrait, E.; Van den Bosh de Aguilar, P.; Bertrand, P. J. Biomed. Mater. Res. 1998, 41, 95. (c) Lhoest, J. B.; Detrait, E.; Van den Bosh de Aguilar, P.; Bertrand, P. J. Biomed. Mater. Res. 1998, 41, 95. (d) Slomkowski, S.; Miksa, B.; Chehimi, M. M.; Delamar, M.; Cabet-Deliry, E.; Majoral, J. P.; Caminade, A. M. React. Funct. Polym. 1999, 41, 45. (e) Coen, M. C.; Lehmann, R.; Groning, P.; Bielmann, M.; Galli, C.; Schlapbach, L. J. Colloid Interface Sci. 2001, 233 (2), 180. (f) Ferrari, S.; Ratner, B. D. Surf. Interface Anal. 2000, 29, 837. (12) Gould, R.; et al. Adv. Chem. Ser. 1964, 43, 1-56. (13) Stenger, D. A.; George, J. H.; Dulcey, C. S.; Hickman, J. J.; Rudolph, A. S.; Nielsen, T. B.; McCort, S. M.; Calvert, J. M. J. Am. Chem. Soc. 1992, 114 (22), 8435.
10.1021/la034870g CCC: $27.50 © 2004 American Chemical Society Published on Web 02/14/2004
Letters
Langmuir, Vol. 20, No. 6, 2004 2055
Figure 1. Adsorption isotherms for XPS data (with standard deviations) based on the nitrogen peak for adsorption of BSA on the hydrophilic (a) and hydrophobic (b) surfaces.
contact angles below 5° were used for hydrophilic surfaces, and those with contact angles above 105° were used for hydrophobic surfaces. BSA (Fisher Biotech, biotech grade) was used in all protein adsorption experiments. The protein adsorption experiments were performed in an 8-mL staining jar in quadruplicate for 2 h at room temperature with agitation. The surfaces were immersed in phosphate buffer saline (PBS) solution (pH ) 7.4) with protein concentrations ranging from 10 to 2000 µg/mL. After adsorption, the samples were removed, rinsed three times with PBS and once with water, and then dried under a stream of nitrogen. Washed and dried samples were examined using a Kratos Analytical AXIS 165 spectrometer utilizing a monochromatic Al KR X-ray source with a pass energy of 40 eV. Survey spectra (0-1000 eV) and high-resolution energy spectra for silicon, oxygen, carbon, and nitrogen were measured for each sample. The intensities of the nitrogen N(1s) peaks at 400 eV were calculated using an internal standard (after the deconvolution and curve fitting peaks were normalized against the intensity of the silicon peak), and the data were averaged for each sample from three different spots. Even though XPS is a high-vacuum technique and the majority of the water is removed from the protein layer before analysis, because we are measuring relative amounts, this should not affect the results. It is possible the water could be removed differently from the protein layer on the two different surfaces, but we consider this highly unlikely. Figure 1a,b represents the adsorption curves from the averaged nitrogen peak XPS data, which is proportional to BSA adsorption, on the hydrophilic and hydrophobic surfaces, respectively. It is apparent from Figure 1 that at pH ) 7.4 much more protein is observed on the hydrophilic surface versus the hydrophobic surface. For the hydrophilic surface, the amount of adsorbed protein increases rapidly with protein concentration in solution before reaching a plateau. For the hydrophobic surface, the amount of adsorbed protein changes only slightly with protein concentration in solution, and at protein concentrations above 0.2 mg/mL,
Figure 2. Adsorption isotherms with calculated K values based on XPS data obtained for a hydrophilic surface (a) and a hydrophobic surface (b).
no further increase in the amount of protein on either surface is observed. To model the protein adsorption, we have used a modified Langmuir adsorption isotherm (eq 1), where Q ) monolayer coverage, K ) binding constant at steady state, and C ) molar concentration.
Q ) KC/(1 + KC)
(1)
We are defining the final state reached as a “reactionsite-limited adsorbed layer” rather than the very ambiguous term “monolayer”.13 Because Q ) N/Nm, where N is the amount of material on the surface at a given concentration and Nm is the amount on the surface at final coverage, eq 1 can be rearranged to
C/N ) C/Nm + 1/KNm
(2)
The surface binding constant can be calculated by plotting C/N versus C. The amount of protein adsorbed on the surface, N, can be determined from XPS analysis using the integrated area of the nitrogen peak or carbon carbonyl peak. Figure 2a represents the adsorption isotherm based on the nitrogen XPS analyses obtained for our representative hydrophilic surface. From the binding isotherm, it is clear that the data are best fit by two linear regions with different slopes. Therefore, the data were fitted for lower and higher protein concentrations using eq 2. The data indicate that at first BSA is adsorbed to the surface with an average binding constant of K1 ) 0.36 (σ ) 0.06). At a certain coverage, however, further BSA adsorption appears to occur via a different mechanism and the second binding constant is lower and equal to K2 ) 3.6 × 10-3 (σ ) 0.9 × 10-3). Figure 2b shows the adsorption isotherm based on data obtained for adsorption of BSA on the hydrophobic surface.
2056
Langmuir, Vol. 20, No. 6, 2004
Figure 3. Changes in water contact angle upon adsorption of BSA on two different surfaces: hydrophilic (diamonds) and hydrophobic (circles).
In this case, the two-step process is not observed, even at very high protein concentrations. The attempt to fit these data separately for lower and higher protein concentration led to the same slope values for both curves within error limits; therefore, a single binding constant of K1 ) 0.06 (σ ) 0.01) was determined. Despite the different amounts of BSA deposited, which apparently proceed by different mechanisms, passivation of the surface is achieved in both cases. At our experimental pH (above the BSA isoelectric point, pI ) 4.3), BSA is charged and may interact more rigorously with the hydrophilic surface than with the hydrophobic surface, which could explain why it proceeds by a different mechanism to the final state. It has been postulated that the amount of adsorbed BSA and the structure of the adsorbed protein layer strongly depend on the material properties of the surface14,15 and that certain properties of an adsorbed protein layer might be dependent on the substrate (adsorbant) itself.16 However, little progress has been made toward determining the mechanism(s) of adsorption or for the method of passivation of a surface by the serum albumins. In an initial attempt to determine the structure of the adsorbed BSA layer, contact angle measurements were performed on the surfaces after protein adsorption. Figure 3 indicates how the contact angle varies with changes in the protein concentration on the two different surfaces used in the experiments just described. Remarkably, on both surfaces, the same ultimate contact angle is reached at maximum surface coverage. On the hydrophilic surface, a gradual increase was observed with increasing protein concentration until a maximum coverage is reached where the final contact angle is Θ ) 68°, σ ) 5 (diamonds curve). On the hydrophobic surface, the wettability of the surface decreased in the presence of
Letters
adsorbed protein (circles curve), ultimately reaching essentially the same value of Θ ) 70°, σ ) 1. Because contact angles are highly sensitive to small changes in the composition of the gaseous, liquid, and solid phases, one can assume that the same wetting ability of the final protein film is indicative of very similar surface properties. On the basis of these contact angle results, we postulate that even though adsorption on two distinctly different surfaces occurs by different steps, when the final state is reached, the outermost layer of protein has the same overall conformation with similar groups extended out from the surface. Unlike previous work that has postulated that there are no thermodynamically stable intermediate states until denaturization,17 we believe this mechanism proceeds through a series of thermodynamically stable intermediates. It has previously been shown that BSA can exist in solution in two states.18 In addition, these intermediates are functional and programmed to passivate all surfaces in a stepwise process such that the final state gives the same outer structure, and this structure prevents further protein adsorption. However, we do not know at this point what those different conformation changes are because XPS will only reveal relative changes in the amount of protein layer thickness and will not provide structural information except in unique experimental situations. In conclusion, we have shown that BSA adsorption on a hydrophilic surface consists of two modes, one that appears to cover the surface very aggressively and a subsequent mode that interacts with this initially deposited layer to prevent further adsorption (i.e., surface passivation). Adsorption on hydrophobic surfaces proceeds in a distinctly different fashion in which surface passivation is reached in a single step. We have determined this by two different methods: XPS analysis and contact angle measurements. We postulate that there is a controlled loss of tertiary structure of BSA in discrete units that allows a specific structure to be formed on the surface, which allows a unique surface to be exposed that inhibits further protein adsorption regardless of the starting surface composition. Clearly, further definition of the structure of the outer layer is very important, and we are attempting to determine that currently. We are continuing these experiments to determine the mechanisms that BSA and other serum albumins utilize to give virtually universal surface passivation to further protein adsorption. Acknowledgment. NSF Grant ECS-0003227 and Grant F30602-01-2-0541 from the Microsystems Technology office at DARPA supported this work. LA034870G
(14) Norde, W.; MacRitchie, F.; Nowicka, G.; Lyklema, J. J. Colloid Interface Sci. 1986, 112, 447. (15) Norde, W.; Giocomelli, C. E. J. Biotechnol. 2000, 259. (16) Zeng, H.; Chittur, K. K.; Lacefield, W. R. Biomaterials 1999, 20, 377.
(17) Norde, W.; Zoungrana, T. Biotechnol. Appl. Biochem. 1998, 28, 133-143. (18) Foster, J. F.; Sogami, M.; Petersen, H. A.; Leonard, W. J., Jr. J. Biol. Chem. 1965, 240 (6), 2495-2502.
