Langmuir 1998, 14, 4535-4544 4535

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 self-
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assembled 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
Publication Date (Web): July 11, 1998 | doi: 10.1021/la970916s

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 to design surfaces that would adsorb albumin quickly and

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 exten-
sively 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: foster@polymer.uakron.edu.
(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.
tenaciously.
One of albumin’s important functions in the blood is to
transport long (C12-C20) unesterified fatty acids.5 There-
fore, 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
 4536 Langmuir, Vol. 14, No. 16, 1998
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 character-
ization of the self-assembled monolayers (SAMs) and
protein layers was performed using the X-ray reflectivity
(XR) technique. XR is extremely sensitive to the thick-
nesses 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
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were only partially covered with albumin molecules.
As in the previous work,10 SAMs of hexadecyltrichlo-
rosilane have been used as the model surfaces. The
application of SAMs for protein adsorption studies is
Publication Date (Web): July 11, 1998 | doi: 10.1021/la970916s
advantageous for several reasons. SAMs are robust in an
aqueous environment, unlikely to rearrange when trans-
ferred 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, sub-
strates 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. Adsorp-
tion 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 fluo-
rescence (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 Langmuir-
Blodgett-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. Root-
mean-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 reflectom-
etry.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

layer Fel/FelSi D, Å σ, Å
Silicon Oxide Substrate
silicon oxide 1.07 ( 0.05 15.0 ( 3.0 2.6 ( 0.4
silicon 1.00 1.7 ( 0.4
Fully Formed SAM Substrate
alkyl chains 0.41 ( 0.03 15.2 ( 2.0 2.3 ( 0.4
interface 0.66 ( 0.05 5.7 ( 1.0 1.3 ( 0.4
silicon oxide 1.02 ( 0.05 15.0 ( 3.0 1.3 ( 0.4
silicon 1.00 1.7 ( 0.4
Partially Formed SAM Substrate
alkyl chains 0.38 ( 0.03 9.6 ( 1.0 2.1 ( 0.4
interface 0.60 ( 0.05 6.0 ( 1.0 1.7 ( 0.4
silicon oxide 1.02 ( 0.05 15.0 ( 3.0 1.3 ( 0.4
silicon 1.00 1.7 ( 0.4

Table 2. Water Contact Angles for Silicon Substrate,

Complete and Partially Formed Alkylsiloxane SAMs
advancing water receding water
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Figure 1. X-ray reflectivity data for the silicon substrate contact angle, deg contact angle, deg
(circles), fully formed SAM substrate (squares), and partially silicon surface fully wettable
formed SAM substrate (diamonds). complete SAM 109 ( 2 90 ( 2
partial SAM 104 ( 2 91 ( 2
profile could be converted into a mass density profile. The
Publication Date (Web): July 11, 1998 | doi: 10.1021/la970916s

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 adsorp-
tion 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, deter-
mined 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.
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 de-
creased 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 Adsorp-
tion. 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 concen-
trations 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.
 4538 Langmuir, Vol. 14, No. 16, 1998 Sheller et al.
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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).
Publication Date (Web): July 11, 1998 | doi: 10.1021/la970916s

Table 3. Model Parameters for the Fits Shown in Figure

3a
layer Fel/FelSi D, Å σ, Å
0.5 mg/mL
protein layer 0.56 ( 0.03 16.0 ( 2.0 3.4 ( 0.9
alkyl chains 0.36 ( 0.03 19.2 ( 2.0 1.3 ( 0.9
interface 0.60 ( 0.05 5.6 ( 1.0 3.4 ( 0.4
silicon oxide 1.02 ( 0.05 15.0 ( 3.0 1.3 ( 0.4
silicon 1.00 1.7 ( 0.4
0.1 mg/mL
protein layer 0.43 ( 0.03 17.0 ( 2.0 4.2 ( 0.9
alkyl chains 0.35 ( 0.03 20.0 ( 2.0 1.3 ( 0.9
interface 0.63 ( 0.05 4.9 ( 1.0 2.6 ( 0.4
silicon oxide 1.02 ( 0.05 15.0 ( 3.0 1.3 ( 0.4
silicon 1.00 1.7 ( 0.4
0.05 mg/mL
protein layer 0.18 ( 0.05 30.0 ( 3.0 7.2 ( 0.9
alkyl chains 0.35 ( 0.05 14.4 ( 3.0 1.7 ( 0.9
interface 0.56 ( 0.05 6.0 ( 2.0 1.3 ( 0.4
silicon oxide 1.02 ( 0.05 15.0 ( 3.0 1.3 ( 0.4
Figure 2. TappingMode AFM images of substrates prior to
silicon 1.00 1.7 ( 0.4
adsorption are nearly featureless and presented here only to
demonstrate the lateral uniformity of the SAM and for 0.01 mg/mL
comparison with the images of adsorbed protein layers: com- protein layer 0.14 ( 0.05 36.0 ( 3.0 6.8 ( 0.9
plete HTS SAM (a), rms roughness 2.2 Å; incomplete HTS SAM alkyl chains 0.36 ( 0.05 15.6 ( 3.0 1.3 ( 0.9
(b), rms roughness 1.1 Å. Roughnesses was measured on an interface 0.56 ( 0.05 5.6 ( 2.0 1.3 ( 0.4
area of 2.5 × 2.5 µm. silicon oxide 1.02 ( 0.05 15.0 ( 3.0 1.3 ( 0.4
silicon 1.00 1.7 ( 0.4
of the protein films. However, since the protein layers a The concentrations in the table are the bulk concentrations of

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 experi-
mental 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
albumin solution to which substrates were exposed.
images by applying a bearing analysis.22 For concentra-
tions 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 Instru-
ments, Inc., Santa Barbara, CA, 1994.
 Albumin Adsorbed on SAMs
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Publication Date (Web): July 11, 1998 | doi: 10.1021/la970916s
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 sub-
strates 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.
Langmuir, Vol. 14, No. 16, 1998 4539
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
 4540 Langmuir, Vol. 14, No. 16, 1998 Sheller et al.

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 after SDS elution
layer Fel/FelSi D, Å σ, Å Fel/FelSi D, Å σ, Å
Bare Silicon Substrate
protein layer 0.42 ( 0.05 11.2 ( 2.0 2.8 ( 0.9
silicon oxide 1.02 ( 0.05 15.0 ( 3.0 2.8 ( 0.4 1.02 ( 0.05 15.0 ( 3.0 3.6 ( 0.9
silicon 1.00 1.7 ( 0.4 1.00 1.7 ( 0.4
Complete SAM Substrate
protein layer 0.43 ( 0.05 17.0 ( 2.0 4.2 ( 0.9
alkyl chains 0.35 ( 0.05 20.0 ( 2.0 1.3 ( 0.9 0.41 ( 0.03 16.8 ( 2.0 2.8 ( 0.9
interface 0.63 ( 0.05 4.9 ( 1.0 2.6 ( 0.4 0.61 ( 0.05 5.1 ( 1.0 1.7 ( 0.4
silicon oxide 1.02 ( 0.05 15.0 ( 3.0 1.3 ( 0.4 1.02 ( 0.05 15.0 ( 3.0 1.3 ( 0.4
silicon 1.00 1.7 ( 0.4 1.00 1.7 ( 0.4
Incomplete SAM Substrate
protein layer 0.57 ( 0.03 12.5 ( 2.0 3.7 ( 0.9 0.48 ( 0.03 9.0 ( 3.0 3.8 ( 0.9
alkyl chains 0.35 ( 0.03 12.5 ( 2.0 1.6 ( 0.9 0.38 ( 0.03 11.0 ( 3.0 3.0 ( 0.9
interface 0.58 ( 0.05 8.0 ( 1.0 2.6 ( 0.4 0.56 ( 0.05 8.0 ( 2.0 3.4 ( 0.4
silicon oxide 1.02 ( 0.05 15.0 ( 3.0 1.7 ( 0.4 1.02 ( 0.05 15.0 ( 3.0 1.7 ( 0.4
silicon 1.00 1.7 ( 0.4 1.00 1.7 ( 0.4
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a 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 layers adsorbed from solution. Local topography data
Therefore, the actual amount of protein surface coverage obtained with AFM were complemented with X-ray
Publication Date (Web): July 11, 1998 | doi: 10.1021/la970916s

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 10-
15%. 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 scat-
tering 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. Never-
theless, 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. Com-
parison 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.
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, con-
formational changes, and desorption of protein mol-
ecules,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 Concen-
tration. 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 concen-
trations 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 concentra-
tion 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 Langmuir, Vol. 14, No. 16, 1998 4541
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Publication Date (Web): July 11, 1998 | doi: 10.1021/la970916s

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 adsorp-
tion (circles); after albumin was adsorbed (squares); after the Figure 6. AFM images of different substrates after adsorption
adsorbed albumin was eluted with SDS (diamonds). of albumin from 0.1 mg/mL solution: (a) bare silicon substrate,
surface roughness 3.7 Å rms; (b) complete HTS monolayer,
by the protein molecule.31-34 By alteration of its dimen- surface roughness 3.2 Å rms; (c) incomplete HTS monolayer,
sions while spreading, a protein molecule achieves closer surface roughness 2.8 Å rms.
contact with the substrate, increasing the number and
strength of the protein-surface interactions. The degree
(31) Norde, W.; MacRitchie, F.; Nowicka, G.; Lyklema, J. J. Colloid of conformational change for each particular protein
Interface Sci. 1986, 112, 447. depends on various factors, such as the rate of protein
 4542 Langmuir, Vol. 14, No. 16, 1998
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Publication Date (Web): July 11, 1998 | doi: 10.1021/la970916s
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 rough-
ness 2.8 Å rms.
Sheller et al.
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 lit-
erature 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 confor-
mational 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,
(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.; Pokorná, Z.; Cepalova, N. A., Moiseev,
Yu. V.; Kálal, 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
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Publication Date (Web): July 11, 1998 | doi: 10.1021/la970916s
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 es-
sentially 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 experi-
mentally 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.
Langmuir, Vol. 14, No. 16, 1998 4543
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 agree-
ment 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 ad-
sorption 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.
 4544 Langmuir, Vol. 14, No. 16, 1998
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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
Publication Date (Web): July 11, 1998 | doi: 10.1021/la970916s
incomplete SAM substrates, respectively. Bulk protein con-
centration 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 diffu-
sion.45 Rabe and Tilton46 have suggested that lateral
diffusion of adsorbed proteins plays a role in albumin
adsorption. Further AFM measurements at low concen-
trations 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 confor-
mation 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.
Sheller et al.
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 ac-
cessible. We cannot dismiss the possibility of the forma-
tion 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 consider-
able 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, “incom-
plete” SAMs resisted SDS elution, which suggests a higher
tenacity of protein adsorption on those surfaces.
Acknowledgment. Research support from The Whi-
taker Foundation is gratefully acknowledged. The authors
wish to thank Dr. L. Lander for providing the hexade-
cyltrichlorosilane 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.