Langmuir 1997, 13, 1881-1883
1881
Variation in Tenacity of Protein Adsorption on Self-Assembled Monolayers with Monolayer Order As Observed by X-ray Reflectivity Stanislaw Petrash, Nina B. Sheller, William Dando, and Mark D. Foster* The University of Akron, Institute of Polymer Science, Akron, Ohio 44325-3909 Received November 6, 1996. In Final Form: February 18, 1997X Differences in the adsorption of human serum albumin (HSA) to hydrophobic self-assembled monolayers of hexadecyltrichlorosilane (HTS) with different layer packings have been observed using X-ray reflectivity. The monolayers with various thicknesses and electron density parameters were prepared using different deposition times. After the monolayers were exposed to 0.1 mg/mL solutions of HSA, a layer of albumin molecules was found adsorbed to the surface. When eluting with sodium dodecyl sulfate (SDS), all adsorbed protein was removed from the more densely packed surfaces. In contrast, a significant fraction of protein molecules adsorbed to less densely packed layers resisted elution with SDS. We conclude that more mobile, less densely packed HTS layers can better accommodate the albumin molecules, which results in more tenacious binding.
Introduction In recent years, self-assembly of organic monolayers has shown great potential as a tool for tailoring surface properties to meet specific goals.1 The robustness of selfassembled monolayers (SAMs) in an aqueous environment, highly uniform surface structure, and relative ease of varying their functionality make SAMs particularly suitable for protein adsorption studies.2,4 Protein adsorption is the first and most important event in the series of processes that take place when a surface is exposed to a biological fluid. Extensive studies have been done to establish the relationships between the physicochemical properties of a given surface, its protein adsorption characteristics and surface biocompatibility.3 Generally, the more hydrophobic the surface, the greater the amount of protein adsorbed on this surface. Results for adsorption of four different proteins onto SAMs of alkanethiolates with terminal groups of different hydrophobicities4 are consistent with the idea that hydrophobic interactions are important in protein adsorption. It has been suggested that selective adsorption of a certain protein in a particular state could be one of the pathways for developing a biocompatible surface.5 Polymer surfaces preadsorbed with human serum albumin (HSA) have been shown to be modestly thromboresistant on a short time scale.6 An apparent problem has been that with time some albumin molecules desorb into solution, diminishing this passivating effect. In this case, general, nonspecific hydrophobic interactions between albumin and the surface appear not to be strong enough to prevent protein desorption. It is well-known that albumin has several strong binding sites for fatty acid molecules.7 Therefore, a polymer surface’s specific affinity to albumin can be increased
significantly by grafting alkyl chains to the surface of the polymer.8 It has been shown that specific interactions between albumin alkyl binding sites and the alkylated polymer surface may be responsible for more tenacious binding that resists elution with sodium dodecyl sulfate9,10 and also for an increased initial adsorption rate of the protein.11 However, surface modification by alkylation of a polymer surface has one significant disadvantage. Studies of environmentally induced restructuring of polymer surfaces suggest that in water, hydrophobic portions of the polymer can migrate away from the solid-liquid interface, making themselves less accessible for albumin.12 This should not be the case with SAMs of organosilanes because the alkyl tails are covalently bonded to the hard crystalline surface of silicon. The lateral density of a SAM can be adjusted by varying the time of self-assembly so that interactions between adjacent alkyl chains will cause them to still extend into the aqueous media, and at the same time the tails will have enough flexibility to interact with the protein molecule. We studied the adsorption of human serum albumin onto the surfaces of HTS monolayers by X-ray reflectometry. The exquisite sensitivity of X-ray reflectometry to electron density profiles of thin films makes it possible to resolve subtle differences in adsorbed protein layers. Measurements of the amount of protein remaining on surfaces after elution with a strong detergent can provide a means for comparing the tenacity of protein molecules binding to different SAM surfaces. Materials and Methods
* To whom correspondence should be addressed. E-mail address:
[email protected]. X Abstract published in Advance ACS Abstracts, March 15, 1997.
Polished single crystal silicon wafers (Semiconductor Processing) were cleaned for about 1 h in a mixture of 30% hydrogen peroxide and 70% sulfuric acid (“piranha” solution) heated to approximately 75 °C. After cleaning, the substrates were rinsed in distilled water and dried with a stream of dry nitrogen. The HTS deposition was performed by immersing a glass substrate
(1) Ulman, A. An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self-Assembly; Academic Press: San Diego, CA, 1991. (2) Fragneto, G.; Thomas, R. K.; Rennie, A. R.; Penfold, J. Science 1995, 267, 657. (3) Sevastianov, V. I. In High Performance Biomaterials: A Comprehensive Guide to Medical/Pharmaceutical Applications; Szycher, M., Ed.; Technomic Press: Lancaster, PA, 1991; Chapter 21. (4) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164. (5) Ratner, B. D. J. Biomed. Mater. Res. 1993, 27, 837. (6) Lyman, D. J.; Knutson, K.; McNeil, B.; Shibatani, K. Trans. Am. Soc. Artif. Intern. Organs 1975, 21, 49. (7) Spector, A. A. J. Lipid. Res. 1975, 16, 165.
(8) Eberhart, R. C.; Munro, M. S.; Frautschi, J. R.; Sevastianov, V. I. In Proteins at Interfaces; Brash, J., Horbett, T., Eds.; ACS Symposium Series 343; American Chemical Society: Washington, DC, 1987; Chapter 24. (9) Riccitelli, S. D.; Schlatterer, R. G.; Hendrix, J. A.; Williams, G. B.; Eberhart, R.C. Trans. Am. Soc. Artif. Intern. Organs 1985, 31, 250. (10) Frautschi, J. R.; Eberhart, R. C. Proceedings of the 5th Southern Biomedical Engineering Conference; Pergamon Press: New York, 1986; p 425. (11) Pitt, W. G.; Cooper, S. L. J. Biomed. Mater. Res. 1988, 22, 359. (12) Ruckenstein, E.; Gourisankar, G. V. J. Colloid. Interface Sci. 1985, 107, 488.
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1882 Langmuir, Vol. 13, No. 7, 1997
Letters
Figure 1. X-ray reflectivity results for HSA adsorption on a complete HTS monolayer.
Figure 2. X-ray reflectivity results for HSA adsorption on a partial HTS monolayer.
into a 1% solution of hexadecyltrichlorosilane in purified hexadecane heated to ∼65 °C on a hot plate. The deposition time was varied from 30 s up to 5 h. Substrates were then removed from the solution and rinsed subsequently in methylene chloride and chloroform. After HTS monolayer deposition, all slides were characterized using the X-ray reflectometry technique.13 X-rays were generated by a rotating anode source (Rigaku, 12 kW, RU 200, Cu KR, λ ) 1.54 Å). A two-axis automated goniometer with a pyrolitic graphite monochromator and slit collimation (δλ/λ ) 0.022; δΘ/Θ ) 0.002) was used to obtain X-ray reflectivity data. The experimental curves were then fit with simulated reflectivity data calculated using an optical matrix formalism. Contact angles for monolayers were determined using a RameHart NRL 100 contact angle goniometer equipped with an environmentally controlled chamber and tilting base. After a drop of water saline solution (0.9% NaCl, Abbot Labs, for injection, NDS 0074-1583-02) was placed on the substrate, it was allowed to equilibrate for 15-30 min in the saturated vapor environment, at 20 °C. Then the goniometer was tilted to an angle of 35o and both advanced and receding contact angles were determined. Human serum albumin (99%, Fraction V, Sigma, essentially fatty acid and globulin free) was used as received. Phosphatebuffered saline (PBS, pH 7.2) was used as a standard dilution medium. Adsorption experiments were performed in a special adsorption cell (volume 50 mL) as follows. Two substrates covered with SAMs were placed vertically in the adsorption cell. One monolayer had been deposited for 5 h and was assumed to be a “complete” SAM. The other SAM was deposited for a shorter time to yield a “partially formed” SAM. The cell was filled with buffer and left alone for 15 min. Then the cell was quickly flushed with 250 mL of 0.1 mg/mL solution of HSA in PBS buffer. After 1 h of adsorption the cell was flushed again with more than 250 mL of protein-free buffer. At this point, if a desorption experiment was performed, the cell was flushed with 250 mL of a 1% solution of sodium dodecyl sulfate (SDS, Sigma) in PBS buffer and left for 1 h. During the entire adsorption/desorption procedure, contact with air was scrupulously avoided. After all manipulations were finished, the cell was opened and the samples were rinsed with distilled, deionized water to remove buffer salts and blow-dried with a stream of dry nitrogen. The samples were then measured immediately with the X-ray reflectometer.
chains) proposed by Tidswell et al.14 For monolayers exposed to the protein solution, another layer representing the adsorbed protein was added to the model; parameters are shown in Table 1. The electron density values Fel are normalized relative to the electron density of silicon FelSi. D is the thickness of the layer, and σ is the rms roughness of the interface between the corresponding layer and the layer on top of it. As deposition time decreases from 5 h to 30 s, the thickness of the alkyl chain layer gradually decreases (intermediate data not shown) from 16.5 to 7.3 Å. This is evident from the fact that the first minimum in the reflectivity curve shifts to higher values of the scattering vector, Q (Figures 1 and 2, top curves). These results suggest that alkyl tails are tilting away from the vertical position due to the decreased lateral density of hydrocarbon chains. The partially formed film remains approximately uniform on a length scale of order 100 Å, because, as was indicated in Wasserman et al.,15 the reflectivity results for an incomplete monolayer are not consistent with a model of partially formed films as islands of close-packed, fully-extended molecules. The electron density model for a film of islands would consist of a layer of the same thickness as that of a complete SAM, but with much lower electron density. This has not been observed in our case (see Table 1). Preliminary AFM results16 also show the partially formed monolayers to be continuous. The contact angle measurement results shown in Table 2 also seem to correspond to increased chain tilt due to higher chain flexibility (lower spatial constraints). The advancing contact angles for the short deposition monolayer are slightly lower than those for the monolayer deposited for a much longer time, suggesting that some of the backbone CH2 groups are exposed along with terminal CH3 groups. Again, contact angle hysteresis values indicate that the hydrophilic surface of the silicon is not exposed, showing that there are no significant defects in the surface covered with HTS. After the previously characterized HTS surfaces are exposed to the albumin solution, X-ray reflectivity data indicate that even after rigorous rinsing with water, there is a layer of protein adsorbed on top of the SAM (middle curves in Figures 1 and 2). This is consistent with the fact that albumin has a high affinity for hydrophobic surfaces. XPS data (not shown) also show the presence
Results and Discussion The X-ray reflectivity data from an HTS monolayer deposited for 5 h (“complete” SAM) along with the corresponding model are shown in Figure 1. The reflectivity data for the monolayer that resulted from the 30 s deposition (“partial” SAM) are shown in Figure 2. All fits for SAMs were obtained using the three-layer model (silicon oxide layer, interface layer, and layer of alkyl (13) Tidswell, I. M.; Ocko, B. M.; Pershan, P. S.; Wasserman, S. R.; Whitesides, G. M.; Axe, J. D. Phys. Rev. B 1990, 41, 1111. Foster, M. D. Crit. Rev. Anal. Chem. 1993, 24, 179.
(14) Tidswell, I. M.; Ocko, B. M.; Pershan, P. S.; Wasserman, S. R.; Whitesides, G. M.; Axe, J. D. Phys. Rev. B 1990, 41, 1111. (15) Wasserman, S. R.; Whitesides, G. M.; Tidswell, I. M.; Ocko, B. M.; Pershan, P. S.; Axe, J. D. J. Am. Chem. Soc. 1989, 111, 5852. (16) Sheller, N. B.; Petrash, S.; Foster, M. D.; Bliznyuk, V.; Tsukruk, V. V. To be published.
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Langmuir, Vol. 13, No. 7, 1997 1883 Table 1. Parameters for the Fits Shown in Figures 1 and 2 complete SAM layer
Fel/FelSi
partial SAM
D, Å
σ, Å
alkyl chains interface silicon oxide silicon
0.43 ( 0.02 0.63 ( 0.05 1.02 ( 0.02 1.00
16.5 ( 0.5 5.4 ( 1.0 15.0 ( 2.0
Before Adsorption 2.4 ( 0.4 1.5 ( 0.2 1.6 ( 0.4 1.7 ( 0.4
protein layer alkyl chains interface20 silicon oxide silicon
0.41 ( 0.02 0.30 ( 0.03 0.48 ( 0.05 1.02 ( 0.02 1.00
14.4 ( 1.0 17.4 ( 1.0 6.4 ( 1.0 15.0 ( 2.0
After Adsorption 3.2 ( 0.4 1.7 ( 0.4 2.1 ( 0.2 1.6 ( 0.4 1.7 ( 0.4
Fel/FelSi
D, Å
σ, Å
0.36 ( 0.02 0.51 ( 0.03 1.02 ( 0.02 1.00
7.3 ( 0.5 6.7 ( 1.0 15.0 ( 2.0
1.3 ( 0.4 0.6 ( 0.2 1.3 ( 0.4 1.3 ( 0.4
0.48 ( 0.02 0.37 ( 0.03 0.48 ( 0.03 1.02 ( 0.02 1.00
17.5 ( 2.0 8.5 ( 2.0 6.5 ( 1.0 15.0 ( 2.0
4.5 ( 0.4 2.8 ( 0.4 1.3 ( 0.2 1.3 ( 0.4 1.3 ( 0.4
0.15 ( 0.02 0.50 ( 0.03 0.51 ( 0.03 1.02 ( 0.02 1.00
12.0 ( 2.0 15.5 ( 2.0 5.0 ( 1.0 15.0 ( 2.0
3.8 ( 0.4 3.0 ( 0.4 0.6 ( 0.2 1.3 ( 0.4 1.3 ( 0.4
After Desorption protein layer alkyl chains interface silicon oxide silicon
0.43 ( 0.02 0.53 ( 0.05 1.02 ( 0.02 1.00
16.3 ( 0.5 5.0 ( 1.0 15.0 ( 2.0
Table 2. Water Contact Angles for Complete and Partially Formed Alkylsiloxane SAMs
complete SAM partial SAM
advancing water contact angle, deg
receding water contact angle, deg
109 ( 2 104 ( 2
90 ( 2 91 ( 2
of protein after adsorption by virtue of the appearance of a nitrogen peak. No nitrogen peak is observed in the XPS spectrum prior to adsorption. The parameters of protein layers adsorbed on the surfaces of both “complete” and “partial” monolayers are similar (Table 1). In both cases thickness values are lower than the native dimension of HSA (38 Å),17 indicating significant denaturation of the albumin molecules. One should notice the higher electron density and thickness values of the albumin layer on the “partial” HTS monolayer. This may indicate a higher affinity of the surface with “looser” alkyl chains for HSA molecules. To test this hypothesis, sodium dodecyl sulfate (SDS) desorption experiments were performed. SDS elutability is a common measure of the strength of protein-surface interactions.18 The X-ray reflectivity data from samples after desorption with SDS are shown in the bottom curves of Figures 1 and 2. The reflectivity of the “complete” SAM after desorption is almost identical to that of the surface before it was exposed to protein. It seems that all of the adsorbed protein was desorbed back to the solution. On the other hand, a significant amount of protein remains on the surface of the HTS monolayer with less dense packing of alkyl chains. XPS studies showed no nitrogen signal from the “complete” SAM surface that was exposed to protein solution and then was eluted with SDS. In contrast, there was a nitrogen peak from the surface of the “partial” SAM after exposing it to albumin and after eluting with SDS. The nitrogen signal was reduced after SDS elution. From the electron density results we can estimate that about 30% of the protein molecules resisted elution with SDS and remained on the surface. The corresponding model parameters (Table 1) show an increased thickness and (17) Carter, D. C.; Ho, J. X. Advances in Protein Chemistry; Academic Press: New York, 1994; Vol. 45, p 153. (18) Bohnert, J. L.; Horbett, T. A. J. Colloid. Interface. Sci. 1986, 111, 363.
2.6 ( 0.4 1.3 ( 0.2 1.6 ( 0.4 1.7 ( 0.4
electron density of the hydrocarbon layer under the protein layer. However, the model parameters for the remaining protein on the “partial” monolayer after SDS desorption should be treated as nominal. Preliminary AFM images16 of the protein layer after SDS desorption show that only a fraction of the HTS monolayer surface is covered with irreversibly adsorbed albumin molecules; i.e. the adsorbed protein layer is patchy. The layer model used to fit the XR experimental data in this case must be viewed as an approximation. The significant differences in SDS elutability of HSA from “complete” and “partial” HTS monolayers cannot be explained by slight differences in hydrophobicity as measured from contact angle experiments. We therefore suggest that the higher flexibility of alkyl chains and/or larger spacing of the chains in the “partial” monolayer allows a wider spectrum of interactions with the protein than does the closely packed “complete” layer. It is not possible to deduce details of the differences in the interactions between the protein and SAM for the “complete” and “partial” layers using only the structural information available here. However, this difference is real. The binding of the protein to the more disordered HTS layers is somehow more tenacious. Recent AFM studies of bovine serum albumin (BSA) adsorption onto patterned alkylsilane monolayers19 show that those protein molecules adsorb from low concentration solution (0.04 µg/mL) only on the regions with more disordered hydrocarbon chains. This finding seems to corroborate the differences in SDS elution seen between our “complete” and “partial” SAMs. Acknowledgment. Research support from The Whitaker Foundation and a Richard Sicka Scholarship for Undergraduates in Polymer Science for W.D. are gratefully acknowledged. The authors wish to thank Lorraine Lander for providing the hexadecyltrichlorosilane. LA9610821 (19) Fang, J.; Knobler, C. M. Langmuir 1996, 12, 1368. (20) The small variations in electron densities of the interfacial layer for complete monolayer samples were necessary to improve the quality of the fits at the highest values of Q. The significance of these changes is unclear. In any case, variation in this parameter hardly impacts the “best fit” values obtained for parameters of the greatest interest here, i.e., adsorbed layer and alkyl chain layer thicknesses and densities.
