Atomic Force Microscopy and X-ray Reflectivity Studies of Albumin

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Langmuir 1998, 14, 4535-4544

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Atomic Force Microscopy and X-ray Reflectivity Studies of Albumin Adsorbed onto Self-Assembled Monolayers of Hexadecyltrichlorosilane N. B. Sheller, S. Petrash, and M. D. Foster* Institute of Polymer Science, The University of Akron, Akron, Ohio 44325

V. V. Tsukruk College of Engineering and Applied Sciences, Western Michigan University, Kalamazoo, Michigan 49008 Received August 13, 1997. In Final Form: May 27, 1998 Atomic force microscopy (AFM) and X-ray reflectivity (XR) have been used together to provide a detailed and direct look at the structure of human serum albumin protein adsorbed onto well-characterized selfassembled monolayer (SAM) surfaces at several protein concentrations. The duration of SAM deposition was also varied to investigate the influence of the density of hydrocarbon chains in the SAM on protein binding tenacity. Concurrent study of adsorption to bare silicon wafers with native oxide surfaces provided a comparison with a hydrophilic surface similar to widely studied glass and quartz surfaces. Both AFM and XR measurements showed that after adsorption, rinsing, and drying, the surfaces of all substrates were covered with no more than a single layer of adsorbed protein. Thin dense protein layers were seen for the substrates exposed to protein concentrations of 0.1 and 0.5 mg/mL. Partial surface coverage by protein aggregates having larger thicknesses was seen for substrates exposed to lower concentrations. The tenacity of the protein adsorption on different substrates was tested by eluting the adsorbed protein with a 1% solution of sodium dodecyl sulfate surfactant. This treatment removed almost all protein from the bare silicon surface and from the fully formed, dense SAMs. A significant amount of adsorbed protein remained on the surface of the less dense, “incomplete” monolayers, suggesting that protein adsorbed more tenaciously on that surface.

Introduction The phenomena that occur at the boundary between the surfaces of solid materials and biological fluids, such as blood, have been attracting scientific interest for many years. To understand the variety of processes happening simultaneously in biological systems or, better yet, to obtain a desirable result from a certain process, one has to use surfaces that are well-defined, stable, and easy to control. One also has to use characterization techniques that are powerful and sensitive to provide relevant information about the system under investigation. One of the first and most important processes that occur when the surface is brought in contact with biological fluid is protein adsorption.1 Over the years, vast amounts of data have been collected regarding the adsorption behavior of different proteins. Among those most extensively studied is serum albumin, since it is the most abundant protein in blood. Albumin is believed to be one of the proteins that affect blood coagulation through its adsorption, and therefore it is extremely important in biomaterials research. For instance, surfaces preadsorbed with albumin have been shown to inhibit thrombus formation.2-4 Therefore, various ways have been sought * To whom correspondence should be addressed. Phone: (330)972-5323. FAX: (330)-972-5290. E-mail: [email protected]. (1) Proteins at interfaces II: Fundamentals and Applications; Brash, J. L., Horbett, T. A., Eds.; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995. Sevastianov, V. I. In High Performance Biomaterials: A Comprehensive Guide to Medical/ Pharmaceutical Applications; Szycher, M., Ed.; Technomic Press: Lancaster, 1991; p 313. (2) Grasel, T. G.; Pierce, J. A.; Cooper, S. L. J. Biomed. Mater. Res. 1987, 21, 815.

to design surfaces that would adsorb albumin quickly and tenaciously. One of albumin’s important functions in the blood is to transport long (C12-C20) unesterified fatty acids.5 Therefore, alkylation of surfaces has been used as a pathway to increase the surface binding of albumin. By attaching alkyl chains to polymer surfaces by various methods, researchers have been able to increase surface affinity toward albumin.6-8 It has also been reported that as the length of the grafted alkyl chain increases from 2 to 18 carbon atoms, the albumin desorption rate decreases.9 The interaction between albumin and an alkylated surface is affected not only by alkyl chain length. Density and ordering of the hydrocarbon chains on the surface also have a significant effect. Recent studies by our group have shown that human serum albumin appears to bind more tenaciously to the surfaces covered with less dense, more disordered alkyl chains.10 After exposure to a solution of sodium dodecyl sulfate, almost all human serum albumin protein (HSA) molecules were eluted from a (3) Munro, M. S.; Eberhart, R. C.; Maki, N. J.; Brink, B. E.; Fry, W. J. J. Am. Soc. Artif. Intern. Organs 1983, 6, 65. (4) Lyman, D. J.; Knutson, K.; McNeil, B.; Shibatani, K. Trans. Am. Soc. Artif. Intern. Organs 1975, 21, 49. (5) Spector, A. A. J. Lipid Res. 1975, 16, 165. (6) Pitt, W. G.; Cooper, S. L. J. Biomed. Mater. Res. 1988, 22, 359. (7) Eberhart, R. C.; Munro, M. S.; Frautschi, J. R.; Sevastianov, V. I. In Proteins at Interfaces; Brash, J. L., Horbett, T. A., Eds.; ACS Symposium Series 343; American Chemical Society: Washington, DC, 1987; Chapter 24. (8) Alvarez, C.; Bertorello, H.; Strumla, M.; Sanchez, E. I. Polymer 1996, 37, 3715. (9) Pitt, W. G.; Grasel, T. G.; Cooper, S. L. Biomaterials 1988, 9, 36. (10) Petrash, S.; Sheller, N. B.; Dando, W.; Foster, M. D. Langmuir 1997, 13, 1881.

S0743-7463(97)00916-5 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/11/1998

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surface with closely packed, extended alkyl chains. On the other hand, roughly 30% of the albumin remained on a surface covered with a less dense, thin, and disordered layer of hexadecyltrichlorosilane (HTS). The characterization of the self-assembled monolayers (SAMs) and protein layers was performed using the X-ray reflectivity (XR) technique. XR is extremely sensitive to the thicknesses and electron densities of self-assembled and protein layers (even for monolayers). However, it cannot provide information about lateral features, since it effectively averages over the structure in the surface plane over several square millimeters. In this report we combine the exquisite thickness sensitivity of XR with lateral information obtained by atomic force microscopy to characterize the layers of human serum albumin adsorbed onto surfaces of self-assembled monolayers with different ordering of the alkyl chains. Atomic force microscopy (AFM) has been shown to be an extremely powerful technique for imaging of biological molecules.11,12 In particular, AFM allowed us to characterize substrates that were only partially covered with albumin molecules. As in the previous work,10 SAMs of hexadecyltrichlorosilane have been used as the model surfaces. The application of SAMs for protein adsorption studies is advantageous for several reasons. SAMs are robust in an aqueous environment, unlikely to rearrange when transferred from air to liquid environment and produce highly defined, smooth surfaces. Also, varying the monolayer deposition time can be used to control the ordering of the hydrocarbon chains. Materials and Methods Preparation of Monolayers. Polished single-crystal silicon wafers (Semiconductor Processing) were cleaned with a mixture of H2O2 (30%) and H2SO4 (70%) (“piranha” solution) heated to 70 °C. The “piranha” solution may react violently with organic materials and must be handled with great care. After cleaning, silicon wafers were stored in distilled, deionized water prior to use. Before SAM deposition or adsorption experiments, substrates were blown dry with a stream of dry grade nitrogen. Prior to use the HTS was filtered and distilled. The purity of final product was 99+% on the basis of the NMR measurements. Self-assembly of HTS molecules was performed from a 1% solution of HTS in purified hexadecane at approximately 65 °C. The time of deposition was varied from 30 s to 5 h. After removal from solution, the substrates were rinsed subsequently in methylene chloride and chloroform. Protein Adsorption Procedure. Human serum albumin (99%, Fraction V, Sigma, essentially fatty acid and globulin free) was used as received. Phosphate-buffered saline (PBS, pH 7.2) was used to prepare solutions of desired concentration. Adsorption experiments were performed in a closed adsorption cell (volume 50 mL) as follows. Two different13 substrates were placed vertically in the adsorption cell. The cell was filled with buffer and left alone for 15 min. Then the cell was quickly flushed with 250 mL of HSA solution in PBS buffer having a particular desired concentration. Kinetic data14 for the albumin adsorption onto HTS monolayers measured using total internal reflection fluorescence (TIRF) have shown that the process of adsorption attained a steady state within half an hour after the surface was exposed to protein solution, so an adsorption time of 1 h was used here. After that time 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 additionally flushed with (11) Hansma, H. G.; Hoh, J. H. Annu. Rev. Biophys. Biomol. Struct. 1994, 23, 115. (12) Firtel, M.; Beveridge, T. J. Micron 1995, 26, 347. (13) Either two monolayers with different deposition time were used or a monolayer and an untreated silicon surface were used for comparison. (14) Tremsina, Y.; Sevastianov, V. I.; Petrash, S.; Dando, W.; Foster, M. D., J. Biomater. Sci., Polym. Ed. 1998, 9, 151.

Sheller et al. 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 blown dry with a stream of dry nitrogen. The samples were then characterized with X-ray reflectivity and AFM. Since characterization of the samples was performed at ambient conditions, we cannot dismiss the influence of exposure of the adsorbed protein layers to air during the measurements. Extra care was taken in the preparation and handling of the samples so that all the samples were treated equally. Therefore, any changes in layer structure due to exposure to air were deemed to be the same from sample to sample. The differences in the layer morphologies observed here were due to the processes that occurred in situ, prior to the removal of the sample from the adsorption cell. Also, because after protein adsorption the cell was flushed with an excess of protein-free buffer, both XR and AFM data characterized only the fraction of albumin molecules that was adsorbed irreversibly. Several adsorption/desorption experiments were performed over a 1 year period, each time producing the same results. The experimental protocol used here also had a certain advantage over those in other studies of dry protein layers, since during the adsorption/desorption procedure contact of the substrates with an air-solution interface was avoided. This eliminated the possibility of LangmuirBlodgett-like deposition of proteins adsorbed on the air-liquid interface. AFM Measurements. AFM measurements were performed on a Dimension 3000 microscope system (Digital Instruments, Inc.) according to established procedures.15,16 Silicon cantilevers were used to acquire topography images in TappingMode at room temperature in ambient conditions. The force exerted on the sample was minimized by adjusting the setpoint. The scanning rate was around 1 Hz. Only a “flattening” procedure was applied to raw images before determining heights from the images. Rootmean-square (rms) roughnesses were measured over 1 µm × 1 µm areas, unless noted otherwise. X-ray Reflectivity Measurements. After HTS monolayer deposition all slides were characterized using X-ray reflectometry.17 X-rays were generated by a rotating anode source (Rigaku, RU 200, 12 kW, Cu KR, λ ) 1.54 Å). Reflectivity curves were measured with a two-axis automated goniometer with pyrolitic graphite monochromator and slit collimation (δλ/λ ) 0.022; δΘ/Θ ) 0.002). The curves were measured so as to intentionally capture “diffuse” scattering close to the specular beam along with specular reflection, since the off-specular scattering was not explicitly considered. The data were, however, corrected for background measured far from the specular reflection. Experimental curves were then fit with simulated reflectivity data calculated using an optical matrix formalism. The model used to fit the experimental curves assumed that the adsorbed protein layer was laterally uniform; that is, there were no “patches” or “islands” of adsorbed protein molecules. As will be seen below from the AFM images, on some samples the protein layers remaining after adsorption were not uniform. In those cases, XR data from such layers could still be fit with the “uniform layer model”, where the aggregates, observed with AFM, were represented by a layer with reduced density and uniform thickness equal to the average height of the adsorbed molecules. However, in such cases, the uncertainty in determining the parameters of the adsorbed layers was significantly higher. From fits of the experimental reflectivity data estimates of the adsorbed amounts of protein were calculated from the XR data by the following method. From fitting the XR curves measured before and after protein adsorption, the scattering length density profiles (SLD) were obtained. Then the SLD profile of just the protein layer was obtained by subtracting the SLD profile of the sample prior to adsorption from the SLD profile of the sample after the protein was adsorbed. Since the chemical composition of the albumin was known, the scattering density (15) Frommer, L. Angew. Chem., Int. Ed. Engl. 1992, 31, 1298. (16) Sarid, D. Scanning Force Microscopy; Oxford University Press: New York, 1991. (17) Foster, M. D. Crit. Rev. Anal. Chem. 1993, 24, 179.

Albumin Adsorbed on SAMs

Langmuir, Vol. 14, No. 16, 1998 4537 Table 1. Model Parameters for Fits Shown in Figure 1 Fel/FelSi

layer

σ, Å

D, Å

silicon oxide silicon

Silicon Oxide Substrate 1.07 ( 0.05 15.0 ( 3.0 1.00

2.6 ( 0.4 1.7 ( 0.4

alkyl chains interface silicon oxide silicon

Fully Formed SAM Substrate 0.41 ( 0.03 15.2 ( 2.0 0.66 ( 0.05 5.7 ( 1.0 1.02 ( 0.05 15.0 ( 3.0 1.00

2.3 ( 0.4 1.3 ( 0.4 1.3 ( 0.4 1.7 ( 0.4

alkyl chains interface silicon oxide silicon

Partially Formed SAM Substrate 0.38 ( 0.03 9.6 ( 1.0 0.60 ( 0.05 6.0 ( 1.0 1.02 ( 0.05 15.0 ( 3.0 1.00

2.1 ( 0.4 1.7 ( 0.4 1.3 ( 0.4 1.7 ( 0.4

Table 2. Water Contact Angles for Silicon Substrate, Complete and Partially Formed Alkylsiloxane SAMs

Figure 1. X-ray reflectivity data for the silicon substrate (circles), fully formed SAM substrate (squares), and partially formed SAM substrate (diamonds). profile could be converted into a mass density profile. The adsorbed amount was then obtained by integrating the mass density profile over depth. The uncertainty in the adsorbed amount was a combination of uncertainties in thickness and scattering length density of adsorbed protein layer, which were determined during the fitting procedure. XPS and Contact Angle Measurements. Survey XPS measurements were performed at the MatNet facility at Case Western Reserve University on samples before and after adsorption using a Physical Electronics ESCA 5600 spectrometer with monochromatized Al KR radiation. A takeoff angle of 45° was used. Contact angles for SAMs were determined as described elsewhere.10

Results Substrate Characterization. Figure 1 shows the X-ray reflectivity results for bare silicon substrate, fully formed SAM deposited for 5 h (designated “complete”) and partially formed SAM deposited for 30 s (designated “incomplete”). All fits for the SAMs were obtained using the three-layer model (silicon oxide layer, interface layer and layer of alkyl chains) proposed by Tidswell et al.18 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. The model parameters used to obtain the fits for the experimental data are listed in Table 1. When fitting the data for the bare silicon substrate, we assumed that it was covered with a thin layer of native oxide (Table 1). The surface roughness of silicon, determined from the X-ray data, was 2.6 ( 0.4 Å rms. The roughness determined from the AFM image of the silicon substrate (not shown) was ∼1.4 Å rms (over an area of 0.25 µm2). The roughnesses determined using these two methods were not expected to agree exactly due to the different ranges of roughness frequencies to which the two techniques were sensitive. After the HTS deposition the substrates were found to be covered with monolayers of HTS molecules. As deposition time was decreased from 5 h to 30 s, the thickness of the alkyl chain layer gradually decreased (XR data for intermediate times not shown) from 15.2 ( 2.0 Å to 9.6 ( 1.0 Å. This was evident from a shifting of (18) Tidswell, I. M.; Ocko, B. M.; Pershan, P. S.; Wasserman, S. R.; Whitesides, G. M.; Axe, J. D. Phys. Rev. B 1990, 41, 1111.

advancing water contact angle, deg silicon surface complete SAM partial SAM

109 ( 2 104 ( 2

receding water contact angle, deg

fully wettable

90 ( 2 91 ( 2

the first minimum in the reflectivity curve to higher values of scattering vector, q (Figure 1, middle and bottom curves). These results suggest that the alkyl tails are tilting or bending away from vertical orientation due to the decreased lateral density of the HTS molecules on the substrate. The AFM images in parts a and b of Figure 2 show both the fully and the partially formed monolayers to be continuous. No holes extending to the silicon substrate were observed at this resolution. These findings are in agreement with previous studies of incomplete alkylsilane SAMs.19 The contact angle measurement data shown in Table 2 also agree with data reported previously by others.20 Concentration Dependence of Albumin Adsorption. Human serum albumin was adsorbed onto the surfaces of complete SAMs from solutions with bulk concentrations of 0.5, 0.1, 0.05, and 0.01 mg/mL. These concentrations were chosen so that the region of the albumin adsorption isotherm where a transition to a plateau occurs would be investigated. It was known from previously reported data21 that for bulk albumin concentrations above 0.1 mg/mL the amount of adsorbed albumin does not increase with concentration; that is, adsorption reaches saturation. Therefore, it was expected that at these concentrations the surfaces would be fully covered with protein. Likewise, concentrations below 0.1 mg/mL should have resulted in lesser amounts of adsorbed proteins, possibly indicating partial surface coverage. The X-ray reflectivity curves for various concentrations are shown in Figure 3. One can notice that the shapes of the reflectivity curves for 0.1 and 0.5 mg/mL are significantly different from those of the reflectivity curves for lower concentrations of 0.05 and 0.01 mg/mL. The best fit model parameters for calculated fits are shown in Table 3. For higher protein concentrations (0.1 and 0.5 mg/mL), the AFM measurements revealed that there were no visible holes in the protein layers. Therefore, it was impossible to measure the thickness of the albumin layers in the AFM tapping mode without significant distortion (19) 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. (20) Wasserman, S. R.; Tao, Y.-T.; Whitesides, G. M. Langmuir 1989, 5, 1074. (21) Van Dulm, P.; Norde, W. J. Colloid Interface Sci. 1983, 91, 248.

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Figure 3. X-ray reflectivity data for substrates with protein layers adsorbed from solutions of different concentrations: 0.5 mg/mL (circles); 0.1 mg/mL (squares); 0.05 mg/mL (diamonds); 0.01 mg/mL (triangles). Table 3. Model Parameters for the Fits Shown in Figure 3a layer

Figure 2. TappingMode AFM images of substrates prior to adsorption are nearly featureless and presented here only to demonstrate the lateral uniformity of the SAM and for comparison with the images of adsorbed protein layers: complete HTS SAM (a), rms roughness 2.2 Å; incomplete HTS SAM (b), rms roughness 1.1 Å. Roughnesses was measured on an area of 2.5 × 2.5 µm.

of the protein films. However, since the protein layers appeared to be continuous, as indicated by the AFM images (Figure 4a,b), the HSA layer parameters obtained from the XR data using a single uniform layer model should be representative of the actual layer dimensions. The layer thicknesses were essentially the same within the experimental uncertainty (16 and 17 Å with roughnesses of 3.4 Å rms and 4.2 Å rms for concentrations 0.5 and 0.1 mg/ mL, respectively) as shown in Table 3. The roughness values agree well with surface roughnesses obtained from AFM data (3.5 and 3.2 Å respectively, see Figure 4a,b). Low scattering length densities and high surface roughnesses obtained from the X-ray reflectivity data for the adsorbed protein layers for lower bulk concentrations suggested that the coverage of surfaces with protein was not uniform; that is, the albumin layers were “patchy”. AFM images provided direct evidence of this (Figure 4c,d). For these concentrations it was possible to measure the average thicknesses of the albumin layers from the AFM

Fel/FelSi

D, Å

σ, Å

protein layer alkyl chains interface silicon oxide silicon

0.5 mg/mL 0.56 ( 0.03 16.0 ( 2.0 0.36 ( 0.03 19.2 ( 2.0 0.60 ( 0.05 5.6 ( 1.0 1.02 ( 0.05 15.0 ( 3.0 1.00

3.4 ( 0.9 1.3 ( 0.9 3.4 ( 0.4 1.3 ( 0.4 1.7 ( 0.4

protein layer alkyl chains interface silicon oxide silicon

0.1 mg/mL 0.43 ( 0.03 17.0 ( 2.0 0.35 ( 0.03 20.0 ( 2.0 0.63 ( 0.05 4.9 ( 1.0 1.02 ( 0.05 15.0 ( 3.0 1.00

4.2 ( 0.9 1.3 ( 0.9 2.6 ( 0.4 1.3 ( 0.4 1.7 ( 0.4

protein layer alkyl chains interface silicon oxide silicon

0.05 mg/mL 0.18 ( 0.05 30.0 ( 3.0 0.35 ( 0.05 14.4 ( 3.0 0.56 ( 0.05 6.0 ( 2.0 1.02 ( 0.05 15.0 ( 3.0 1.00

7.2 ( 0.9 1.7 ( 0.9 1.3 ( 0.4 1.3 ( 0.4 1.7 ( 0.4

protein layer alkyl chains interface silicon oxide silicon

0.01 mg/mL 0.14 ( 0.05 36.0 ( 3.0 0.36 ( 0.05 15.6 ( 3.0 0.56 ( 0.05 5.6 ( 2.0 1.02 ( 0.05 15.0 ( 3.0 1.00

6.8 ( 0.9 1.3 ( 0.9 1.3 ( 0.4 1.3 ( 0.4 1.7 ( 0.4

a The concentrations in the table are the bulk concentrations of albumin solution to which substrates were exposed.

images by applying a bearing analysis.22 For concentrations 0.05 and 0.01 mg/mL the thicknesses were 37 and 36 Å, respectively. The thicknesses derived from the XR data were close to those obtained by AFM (30 and 36 Å for 0.05 and 0.01 mg/mL, correspondingly). In these two cases, the values from XR were less precise, since the uniform layer model did not satisfactorily represent the actual “patchy” protein layers. However, in any case the XR data could not be fitted with a model having an albumin layer thickness less than 30 Å. Substrate Dependence of Albumin Adsorption and Desorption. To evaluate the tenacity of albumin adsorption to the surfaces of various substrates, layers (22) Dimension 3000 Microscope Instruction Manual, Digital Instruments, Inc., Santa Barbara, CA, 1994.

Albumin Adsorbed on SAMs

Langmuir, Vol. 14, No. 16, 1998 4539

Figure 4. AFM images of substrates covered with HTS SAMs with albumin layers adsorbed at different bulk protein concentrations: (a) 0.5 mg/mL; (b) 0.1 mg/mL; (c) 0.05 mg/mL; (d) 0.01 mg/mL. Note that the z-range in images a and b is half that in images c and d.

adsorbed from 0.1 mg/mL protein solution were subjected to elution by a solution of sodium dodecyl sulfate (SDS). In Figure 5 the X-ray data for three different substrates are shown: bare silicon (Figure 5a), complete HTS monolayer (Figure 5b) and incomplete HTS monolayer (Figure 5c). The top curves are for the substrates prior to protein adsorption,23 the middle curves are for substrates after albumin was adsorbed on them, and the bottom curves are for the substrates after SDS elution. The model parameters used to obtain the fits shown are listed in Table 4. The XR data show that after exposure to 0.1 mg/mL albumin solution for 1 h each of the substrates becomes covered with a dense, uniform layer of protein. The AFM images of these substrates with protein layers are shown in Figure 6. Since the protein layers appear to be laterally homogeneous with no holes, the adsorbed layer parameters can be reliably obtained from XR data (see Table 4). After SDS elution, essentially all the protein is gone from the surface of silicon. The XR curve for the silicon (23) The XR data for substrates prior to adsorption (top curves) are repeated here from Figure 1 for convenience of comparison.

substrate after desorption is similar to the one measured before albumin adsorption. The AFM image (Figure 7a) shows that only several HSA molecules remain on the surface of silicon. There are too few of them to be detected by XR. A little more protein remains on the surface of the complete SAM, as one can see from Figure 7b. At first glance there seems to be a significant amount of protein left on the SAM surface, so one might expect that the XR curve from this sample should differ significantly from the curve for the SAM before protein adsorption. As one can see from the XR data (Figure 5b, bottom curve vs top one), this is not the case. One has to keep in mind that when surface features, such as those seen in Figure 7b, are imaged using the AFM technique, the observed lateral dimensions are significantly larger than the real ones. This broadening of the lateral features of the image is due to the scanning of small surface features with an AFM probe having a finite tip radius. The end radii of the tips used here were ∼10 nm (as reported by the manufacturer). Taking into account that measured topographical heights of the aggregates were 1-3 nm, the lateral dimensions of

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Table 4. Model Parameters Used To Fit the XR Data for Substrates with Adsorbed and Eluted Layers of Albumin (Figure 5a-c; middle and bottom curves)a after albumin adsorption layer

a

Fel/FelSi

D, Å

after SDS elution σ, Å

Fel/FelSi

D, Å

σ, Å

1.02 ( 0.05 1.00

15.0 ( 3.0

3.6 ( 0.9 1.7 ( 0.4

protein layer silicon oxide silicon

0.42 ( 0.05 1.02 ( 0.05 1.00

Bare Silicon Substrate 11.2 ( 2.0 2.8 ( 0.9 15.0 ( 3.0 2.8 ( 0.4 1.7 ( 0.4

protein layer alkyl chains interface silicon oxide silicon

0.43 ( 0.05 0.35 ( 0.05 0.63 ( 0.05 1.02 ( 0.05 1.00

Complete SAM Substrate 17.0 ( 2.0 4.2 ( 0.9 20.0 ( 2.0 1.3 ( 0.9 4.9 ( 1.0 2.6 ( 0.4 15.0 ( 3.0 1.3 ( 0.4 1.7 ( 0.4

0.41 ( 0.03 0.61 ( 0.05 1.02 ( 0.05 1.00

16.8 ( 2.0 5.1 ( 1.0 15.0 ( 3.0

2.8 ( 0.9 1.7 ( 0.4 1.3 ( 0.4 1.7 ( 0.4

protein layer alkyl chains interface silicon oxide silicon

0.57 ( 0.03 0.35 ( 0.03 0.58 ( 0.05 1.02 ( 0.05 1.00

Incomplete SAM Substrate 12.5 ( 2.0 3.7 ( 0.9 12.5 ( 2.0 1.6 ( 0.9 8.0 ( 1.0 2.6 ( 0.4 15.0 ( 3.0 1.7 ( 0.4 1.7 ( 0.4

0.48 ( 0.03 0.38 ( 0.03 0.56 ( 0.05 1.02 ( 0.05 1.00

9.0 ( 3.0 11.0 ( 3.0 8.0 ( 2.0 15.0 ( 3.0

3.8 ( 0.9 3.0 ( 0.9 3.4 ( 0.4 1.7 ( 0.4 1.7 ( 0.4

The fit parameters for the substrates prior to the protein adsorption (top curves) are listed in Table 1.

the imaged features would be extended by up to 14 nm.24,25 Therefore, the actual amount of protein surface coverage is much smaller than that suggested by casual inspection of Figure 7b. Grain size analysis22 of the AFM images yields values for surface coverage on the order of 1015%. If we take into account the effect of broadening of surface features by the tip, the actual surface coverage will be approximately 3-4%. When studied with XR, this kind of surface coverage leads to increased diffuse scattering near the specular beam. However, since the diffuse off-specular scattering was not explicitly separated in the measurements made here, that difference is not evident in the reflectivity curves. Survey XPS measurements from a similarly treated sample (not shown) exhibited no significant nitrogen peaks, suggesting that the amount of protein remaining was below the XPS detection limit. This detection limit was determined by the overall sample volume probed, not simply the ratio of area of protein to total area. Thus, even in the case of a full protein monolayer the measured nitrogen signal in the XPS spectrum yielded a calculated nitrogen composition of only 4 atom %. A comparably large fraction of HSA remains on the surface of the incomplete HTS monolayer. As can be seen in Figure 5c (bottom curve), the uniform layer model cannot fit the corresponding XR data very well. Nevertheless, the XR curve measured after SDS desorption shows that the minimum remains shifted to lower values of q (as compared to the reflectivity from the SAM alone), which clearly indicates that a significant amount of albumin remains on top of the incomplete HTS. Comparison of the AFM images in Figure 6c and Figure 7c also indicates that many of the albumin molecules have remained adsorbed on the partially formed monolayer. The XPS spectrum (not shown) exhibited a nitrogen peak from protein material that resisted desorption. However, its intensity is somewhat less than that for the substrate with protein before the SDS elution. Discussion The primary objective of this work was to use the AFM technique to observe directly the morphology of albumin (24) Quist, A. P.; Bjorck, L. P.; Reimann, C. T.; Oscarsson, S. O.; Sundqvist, B. U. R. Surf. Sci. 1995, 325, L406. (25) Allen, M. J.; Hud, M.; Balooch, R. J.; Tench, W. J.; Siekhaus, W. J.; Balhorn, R. Ultramicroscopy 1992, 42-44, 1095.

layers adsorbed from solution. Local topography data obtained with AFM were complemented with X-ray reflectometry thickness measurements, which averaged over large areas. We believe that our observations are consistent with theoretical descriptions of the various simultaneously occurring processes of adsorption, conformational changes, and desorption of protein molecules,26 which have appeared in the literature,27-29 and we consider our experimental observations in light of those concepts. First we discuss the variation in adsorbed layer morphology with concentration. Then we look at the variation of adsorption tenacity with surface properties. Variation of Albumin Adsorption with Concentration. As bulk protein concentration increases, we observe an increase in the amount of adsorbed albumin. The values for adsorbed amount of albumin, obtained from X-ray measurements, were 0.09 ( 0.03, 0.09 ( 0.03, 0.16 ( 0.02, and 0.18 ( 0.02 µg/cm2 for protein bulk concentrations of 0.01, 0.05, 0.1, and 0.5 mg/mL, respectively. The value of 0.18 ( 0.02 µg/cm2 for the highest concentration of 0.5 mg/mL agrees quite well with the isotherm plateau value of 0.2 µg/cm2 obtained by Van Dulm and Norde21 using iodine-125 labeling for irreversibly adsorbed albumin after 18 h of adsorption on glass substrates made hydrophobic through a surface treatment. Very similar results have been reported by Baszkin and Boissonnade30 for 20 h of HSA adsorption on a polyethylene surface. Such agreement of our results for adsorbed amount with literature data would suggest that the structure observed in the present work corresponds to the steady-state adsorption and that we characterized the adsorbed layer after most of the molecular rearrangements of albumin have taken place. It is well-known that after adsorption proteins such as albumin undergo significant conformational changes, which usually leads to an increase in the area occupied (26) Sevastianov, V. I.; Tremsina, Y. S.; Eberhart, R. C.; Kim, S. W. In Proteins at Interfaces II; Horbett, T. A., Brash, J. L., Eds.; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995; Chapter 14. (27) Sevastianov, V. I.; Belomestnaya, Z. M.; Zimin, N. K. Artif. Organs 1983, 7, 126. (28) Kurrat, R.; Ramsden, J. J.; Prenosil, J. E. J. Chem. Soc., Faraday Trans. 1994, 90, 587. (29) Schaaf, P.; Talbot, J. J. Chem. Phys. 1989, 91, 4401. (30) Baszkin, A.; Boissonnade, M. M. In Proteins at Interfaces II; Horbett, T. A., Brash, J. L., Eds.; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995; Chapter 15.

Albumin Adsorbed on SAMs

Figure 5. (a) X-ray reflectivity data for a bare silicon substrate at different stages of the adsorption-desorption procedure: before the albumin adsorption (circles); after albumin was adsorbed (squares); after the adsorbed albumin was eluted with SDS (diamonds). (b) X-ray reflectivity data for the fully formed SAM substrates at different stages of the adsorption-desorption procedure: before the albumin adsorption (circles); after albumin was adsorbed (squares); after the adsorbed albumin was eluted with SDS (diamonds). (c) X-ray reflectivity data for the partially formed SAM substrates at different stages of adsorption-desorption procedure: before the albumin adsorption (circles); after albumin was adsorbed (squares); after the adsorbed albumin was eluted with SDS (diamonds).

by the protein molecule.31-34 By alteration of its dimensions while spreading, a protein molecule achieves closer contact with the substrate, increasing the number and (31) Norde, W.; MacRitchie, F.; Nowicka, G.; Lyklema, J. J. Colloid Interface Sci. 1986, 112, 447.

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Figure 6. AFM images of different substrates after adsorption of albumin from 0.1 mg/mL solution: (a) bare silicon substrate, surface roughness 3.7 Å rms; (b) complete HTS monolayer, surface roughness 3.2 Å rms; (c) incomplete HTS monolayer, surface roughness 2.8 Å rms.

strength of the protein-surface interactions. The degree of conformational change for each particular protein depends on various factors, such as the rate of protein

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arrival at the interface, the initial orientation of the molecule upon collision with the surface, the time the protein has spent on the surface, and the distance to its nearest neighbors. At high bulk concentrations the rate at which albumin molecules collide with the surface is high, with many proteins in various orientations arriving at the surface simultaneously with their nearest neighbors, resulting in the high surface coverage. We imagine that the side-on conformation is more favorable, and after adsorption these side-on oriented molecules may rearrange (unfold) and further optimize their hold on the surface. Such molecules would be less likely to desorb,35 and in the process of relaxation they can displace the other less deformed, weakly adsorbed protein molecules.26,36 This ensuing competition for adsorption sites leads to the easy displacement of molecules that had initially adsorbed with less favorable orientations. The mechanism of this displacement has been previously described in the literature and is summarized briefly below. It has been commonly observed37,38 that desorption of a single protein as an isolated event is very slow or even nonexistent, but protein exchange (desorption followed by readsorption) is very rapid. Studies performed by Jennissen39 and an extensive review by Andrade and Hlady40 discuss this phenomenon in detail. They argue that, statistically, now and then a protein can break a single bond with a binding site (lift a “foot” from the surface). This bond can then be reestablished and the protein will remain on the surface. In order for a single adsorbed protein to desorb back into the solution this molecule needs to break all the protein-surface bonds (lift all “feet”) almost simultaneously, which is statistically improbable. Now consider the case when many protein molecules are present in the local environment, diffusing and colliding with a surface. In the process of conformational changes upon adsorption, one of these proteins can statistically place a “foot” on the space vacated by the desorbing “foot” of an neighboring adsorbed protein. Therefore, one protein can essentially be lifted off by a number of spreading neighbor proteins. Such spreading of proteins on the surface will also prevent other molecules from adsorbing, leading to a decrease in the rate of adsorption. On the other hand, the corresponding rate of desorption increases, as molecules that are not in favorable orientations are desorbed. As a result, the mass of protein at the surface actually decreases somewhat over time, which results in the appearance of a maximum in adsorbed amount of protein in the kinetic curve (“overshoot” behavior). This “overshoot” has in fact been observed for HSA adsorbing to an HTS layer in kinetic measurements done by Tremsina et al.14 Although adsorption in these measurements was performed under flow, while in the current work adsorption was done under static conditions,

Figure 7. AFM images of albumin layers on various substrates after SDS elution: (a) HSA after elution from silicon substrate, single white spots are believed to be single albumin molecules, surface roughness 1.7 Å rms.; (b) HSA after elution from complete HTS monolayer, surface roughness 7.8 Å rms; (c) HSA after elution from incomplete HTS monolayer, surface roughness 2.8 Å rms.

(32) Garrison, M. D.; Iuliano, D. J.; Saavedra, S. S.; Truskey, G. A.; Reichert, W. M. J. Colloid Interface Sci. 1992, 148, 415. (33) Lenk, T. J.; Ratner, B. D.; Gendreau, R. M.; Chittur, K. J. Biomed. Mater. Res. 1989, 23, 549. (34) Castillo, E. J.; Koenig, J. L.; Anderson, J. M.; Lo, J. Biomaterials 1984, 5, 319. (35) Norde, W.; Haynes, C. A. In Proteins at Interfaces II; Horbett, T. A., Brash, J. L., Eds.; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995; Chapter 2. (36) Tremsina, Yu. S.; Sevastianov, V. I. Biomater.-Living System Interactions 1995, 3, 103. (37) Chan, B. M. C.; Brash, J. L. J. Colloid Interface Sci. 1981, 82, 217. (38) Brynda, E., Houska, M.; Pokorna´, Z.; Cepalova, N. A., Moiseev, Yu. V.; Ka´lal, J. Bioeng. 1978, 2, 411. (39) Jennissen, H. P. J. Chromatogr. 1978, 159, 71. Jennissen, H. P. Z. Physiol. Chem. 1976, 357, 1727. Jennissen, H. P.; Botzet, G. Int. J. Biol. Macromol. 1979, 1, 171. (40) Andrade, J. D.; Hlady, V. Adv. Polym. Sci. 1986, 79, 1.

Albumin Adsorbed on SAMs

Figure 8. Cross sections of AFM images of albumin layers adsorbed onto HTS SAMs from (a) 0.5 mg/mL and (b) 0.01 mg/ mL bulk protein concentration.

a number of literature data suggest that we can still rationalize our observations using essentially the same reasoning that we have used to explain the kinetic results in our previous work. For example, one of the early albumin adsorption experiments performed by Brynda et al.38 compared albumin adsorption on polyethylene under various flow rates as well as under static conditions. The qualitative behavior of the adsorption curves was essentially the same with or without flow, although the adsorption time frame in flow was somewhat different from that under static conditions. The authors concluded that the increase in the flow rate did not change the mechanism of albumin adsorption, but merely accelerated the diffusion of proteins to the adsorption sites due to a decrease in the thickness of the diffusion (boundary) layer near the surface of substrate. Furthermore, “overshoot” behavior like that in our work was observed by Soderquist et al.41 who studied the adsorption of bovine serum albumin on glass beads covered with silicone under static conditions very similar to those used here. They also explained their results in terms of conformational changes in adsorbed albumin molecules, leading to desorption of less optimally adsorbed proteins. Extensive desorption of weakly adsorbed proteins should lead to the formation of a layer that consists mostly of albumin molecules that are highly irreversibly adsorbed because of significant conformational changes in their structure. Thus the thickness of the adsorbed layer would be expected to be less than the thickness of the native molecule. Indeed, in this work we have shown experimentally that at higher concentrations (0.1 and 0.5 mg/ mL) the adsorbed layer has a thickness of about 16-17 Å. This is less than the native thickness of the HSA molecule of 38 Å, suggesting a rather high degree of protein unfolding. A typical AFM cross section of such an albumin layer (Figure 8a) shows that the height distribution is relatively uniform. Let us now consider the adsorbed layer morphology at lower concentration. First we note that the adsorbed (41) Soderquist, M. E.; Walton, A. G. J. Colloid Interface Sci. 1980, 75, 386.

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amounts deduced from fitting the X-ray data are 0.09 ( 0.03 µg/cm2 for both 0.01 and 0.05 mg/mL bulk solution concentrations. A smaller amount of adsorbed protein observed at concentrations below 0.1 mg/mL is in agreement with published isotherms for HSA adsorbed to hydrophobic surfaces.21 At this juncture we note that the protocol used in the present measurements allowed us to characterize the amount of irreversibly adsorbed albumin molecules, that is, proteins that were adsorbed sufficiently strongly to withstand rinsing. At infinitely long adsorption times the saturation value of irreversibly adsorbed protein is determined only by the effective number of active sites on the surface, independent of the concentration of protein in solution. Indeed, very long (several days up to 1 week) adsorption time studies indicate that the amount of irreversibly adsorbed albumin slowly and gradually increases,42 until the surface is saturated with irreversibly adsorbed proteins and the adsorbed amount does not depend on concentration.38 However, an overwhelming majority of experimental data exhibit a concentration dependence in the albumin adsorption isotherms similar to ours. It has been emphasized in the literature41 that such a discrepancy in observations is due to the fact that conformational changes of adsorbed proteins occur over time and that the adsorption process for such proteins as albumin usually occurs in three stages.43 At very short times adsorption rate is diffusion-controlled. Proteins have insufficient time to conformationally adjust to the surface, and adsorption can be reversible. At longer times the conformational changes become important and adsorption becomes only partially reversible. At very long contact times all conformational changes are complete and the adsorption is fully irreversible. As in the many other protein adsorption studies, in the present work the experimental conditions were chosen so that albumin adsorption would be investigated in the second stage, to observe the influence of conformational changes on the morphology of adsorbed protein layers. At lower solution concentrations the rate of arrival of proteins at the surface is low. Therefore adsorption rate is diffusion-limited. There is no intense competition at the surface, since the proteins are much less likely to land in close vicinity to each other in the short period of time when most of the initial spreading of the molecules occurs. Weakly adsorbed proteins that arrive at the surface in less favorable orientations are less likely to be desorbed by more actively spreading neighbors, who happened to adsorb in favorable configurations. As a result, molecules that have arrived in a variety of orientations, persist on the surface. This variety is suggested by the differences in height of molecular aggregates seen in the AFM cross section in Figure 8b. The heights vary from 30 to 80 Å, which is consistent with the smaller and larger dimensions of the native albumin molecule. (The albumin shape can be approximated as a heart-shaped triangle with sides of 80 Å and thickness of 38 Å.44) It is still true that molecules which arrive in the most favorable orientation tend to rearrange and displace neighbors, but many molecules persist for times in less favorable (less tightly adsorbed) states. These times are long on the scale of the kinetics in the concentrated solutions. When the adsorbed layers are rinsed before study, most loosely, reversibly adsorbed (42) Bohnert, J.; Horbett, T. J. Colloid Interface Sci. 1986, 111, 363. (43) Andrade, J. D. In Surface and Interfacial Aspects of Biomedical Polymers. Protein Adsorption; Andrade, J. D., Ed.; Plenum Press: New York, 1985; Vol. 2, p 1. (44) Carter, D. C.; Ho, J. X. Advances in Protein Chemistry; Academic Press: New York, 1994; Vol. 45, p 153.

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Figure 9. Protein mass density profiles calculated from the electron density profiles of corresponding substrates before and after adsorption. The profiles are shifted along the abscissa axis for clarity. The adsorbed amounts are 0.08 ( 0.02, 0.16 ( 0.02, and 0.16 ( 0.02 µg/cm2 for silicon, complete SAM, and incomplete SAM substrates, respectively. Bulk protein concentration is 0.1 mg/mL.

protein may be rinsed away, exposing some surface. However, even allowing for the displacement of some protein before imaging leaves open the question of why the morphology should appear fractal like. This kind of structure is reminiscent of that seen in systems in which morphologies develop by two-dimensional surface diffusion.45 Rabe and Tilton46 have suggested that lateral diffusion of adsorbed proteins plays a role in albumin adsorption. Further AFM measurements at low concentrations including measurements in situ will be required to more fully characterize these structures. Influence of Surface Properties on Adsorbed Protein Layers. Figure 9 shows the mass density profiles of albumin layers adsorbed on three different substrates obtained from the X-ray reflectivity data. Different surface properties clearly result in differently adsorbed protein layers. As should be expected, the amount of irreversibly adsorbed protein is higher on the hydrophobic surfaces of self-assembled monolayers than on the hydrophilic silicon surface.47 The thickness of the albumin layer adsorbed on the incomplete SAM is lower than that on the complete one. This suggests that protein molecules undergo more significant conformational changes in the 1 h adsorption time on the surfaces with a less dense layer of alkyl chains. The very low thickness of the protein layer adsorbed on the hydrophilic bare silicon substrate suggests that in the absence of hydrophobic interactions only those protein molecules that dramatically altered their native conformation were able to adsorb irreversibly. The fact that the SDS surfactant almost completely eluted the albumin (45) Mandelbrot, B. Fractal Geometry of Nature; Freeman: San Francisco, 1982. (46) Rabe, T. E.; Tilton, R. J. Colloid Interface Sci. 1993, 159, 243. (47) Norde, W. In Adhesion and Adsorption of Polymer; Lee, L.-H. Y., Ed.; Plenum Press: New York, 1980; Vol. 2, p 801.

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from the silicon surface suggests that protein-surface hydrophobic interactions are important for tenacious adsorption. A possible explanation for the higher tenacity of the albumin adsorption to the partially formed SAM is that a more flexible surface may conform better to the structure of albumin molecule, resulting in closer contact between the sorbent and the adsorbed protein. Since the lateral density of HTS molecules in the incomplete SAM is less than that in the complete one, the alkyl tails have more freedom to accommodate the albumin molecule, especially its nonpolar regions. Recently, in situ neutron reflectivity studies by our group48 showed evidence that hydrocarbon chains spaced farther apart penetrate inside the HSA layer, inducing bigger changes in conformation, which could make the internal hydrophobic pockets more accessible. We cannot dismiss the possibility of the formation of specific interactions of the alkyl chains with the albumin’s fatty acid binding sites.6 Unfortunately, at this point we cannot deduce the particular details of the protein-SAM interaction using the structural information presented here. Nevertheless, the combination of the X-ray reflectivity and AFM techniques provides considerable information on the morphology of unlabeled protein adsorbed on well-defined model substrates. Conclusion We have studied layers of human serum albumin adsorbed onto surfaces of self-assembled monolayers of hexadecyltrichlorosilanes by TappingMode AFM and X-ray reflectometry. Changes in bulk concentration of protein in solution resulted in different morphologies of the adsorbed albumin layers as observable with the protocol used. Higher bulk concentrations produced dense and thin layers of protein. Partial surface coverage and the formation of protein aggregates with nonuniform height distribution were seen for adsorption at lower concentrations. We attribute those differences to the degree of competition between the adsorbing albumin molecules, which increases with bulk concentration. AFM images show directly that the proteins adsorbed to the partially formed, “incomplete” monolayers resist elution more effectively, further supporting conclusions first suggested by earlier reflectometry results.10 After the surfaces of silicon oxide covered with adsorbed protein layers were exposed to a solution of sodium dodecyl sulfate, AFM observation found that only a few protein molecules resisted elution. A vast majority of the adsorbed protein was also removed from the dense, “complete” monolayers by SDS treatment. On the other hand, a substantial amount of the protein adsorbed onto less dense, “incomplete” SAMs resisted SDS elution, which suggests a higher tenacity of protein adsorption on those surfaces. Acknowledgment. Research support from The Whitaker Foundation is gratefully acknowledged. The authors wish to thank Dr. L. Lander for providing the hexadecyltrichlorosilane and Dr. V. Bliznyuk for technical assistance. LA970916S (48) Petrash, S.; Sheller, N. B.; Foster, M. D.; Bin, Z.; Brittain, W. J.; Majkrzak, C. F. In preparation.