Colloids and Surfaces A: Physicochem. Eng.

Aspects 360 (2010) 210–219

Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical and

Engineering Aspects
journal homepage: www.elsevier.com/locate/colsurfa

Adsorption of human serum albumin (HSA) on modified PET films monitored by

QCM-D, XPS and AFM
Tea Indest a,∗,1 , Janne Laine b , Karin Stana Kleinschek a,1 , Lidija Fras Zemljič a,1
a
Laboratory for Characterization and Processing of Polymers, Faculty of Mechanical Engineering, University of Maribor, Smetanova 17, SI-2000 Maribor, Slovenia
b
Laboratory of Forest Products Chemistry, Helsinki University of Technology, P.O. Box 6300, FI-02015 TKK, Finland

a r t i c l e i n f o a b s t r a c t

Article history: The adsorption behavior of human serum albumin (HSA) on differently modified poly(ethylene tereph-
Received 4 December 2009 thalate) (PET) model film surfaces was studied using the quartz crystal microbalance (QCM), X-ray
Received in revised form 23 February 2010 photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) techniques. The aim was to eval-
Accepted 1 March 2010
uate the influence of different modifications of PET surfaces with selected polysaccharides (chitosan,
Available online 7 March 2010
fucoidan, chitosan sulfate) on HSA adsorption.
As a first step, PET hydrophilicity was increased by alkaline hydrolysis. From these foils model PET
Keywords:
films were prepared by the spin coating technique on a quartz crystal. Selected polysaccharides (chitosan,
QCM-D
PET films
fucoidan and chitosan sulfate) were adsorbed from aqueous solutions on the PET surfaces. Adsorption
Human serum albumin (HSA) of HSA on the films was monitored with QCM-D. The surface composition and morphology of the HSA
Chitosan sulfate covered PET films was analyzed using XPS and AFM.
Fucoidan It was found that due to steric repulsion the chitosan, fucoidan and chitosan sulfate adlayers reduced
HSA adsorption, especially in case of chitosan/fucoidan and chitosan/chitosan sulfate covered films.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction Thus, protein adsorption will have an influence on the subsequent

Protein adsorption plays a major role in a variety of important
technologically and biologically related processes. For blood-
contact applications, biocompatibility is determined largely by
specific interactions with blood and its components. The surface
structure of any biomaterial governs the biological response, which
is initially determined by protein and cell interactions. There-
fore biomaterials are required to eliminate, or largely reduce,
the non-specific adsorption of blood proteins, in order to avoid
immunological response like for example, surface-induced throm-
bosis [1–3].
Thus it is understandable that nowadays the general aim is to
optimize the cell–polymer interactions by modifying polymer sur-
faces in contact with blood [4]. Various polymeric materials have
been modified by water soluble polymers, such as poly(ethylene
glycol) (PEG), for biomedical application, because they can pre-
vent plasma protein adsorption, platelet adhesion and thrombus
formation [5].
The protein adsorption event occurs well before blood cells
arrive at the biomaterials surface. Therefore, cells primarily contact
a protein layer, rather than the actual surface of the biomaterial.
∗ Corresponding author. Tel.: +386 2 251 6091; fax: +386 2 251 6092.
E-mail address: tea.indest@gmail.com (T. Indest).
1
Member of the European Polysaccharide Network of Excellence (EPNOE).
cellular interactions with foreign surfaces [6–9]. Protein adsorption
at the solid/liquid interface is regulated by numerous factors like
chemical composition, hydrophobicity, pH, surface roughness, etc.,
and this diversity makes it a very complex process [10].
Human serum albumin (HSA) accounts for approximately 50%
of the proteins found in human plasma [11]. Due to its abundance
(≈35–52 g/L in biological fluids such as blood) it is the first protein
involved in the cascade adsorption of proteins on solid surfaces
immersed in a biological medium, and it plays a critical role in
the following evolution of the adsorption processes [6]. The HSA
molecule is a globular, extracellular protein with reported isoelec-
tric point ranging from 4.7 to 5.2 [12,13] and a Mw 65 kDa [14].
Albumin is one of the smallest plasma proteins and, given its highly
polar nature, dissolves easily in water [15]. At pH 7.4, it is an anion
with 200 negative charges per molecule; this gives it a vast capacity
for nonselective binding of many ligands [11]. Albumin has an ellip-
soidal shape, which means that it does not increase the viscosity of
the plasma as much as an elongated molecule such as fibrinogen
does [15].
In haemocompatibility studies, the adsorption of plasma pro-
teins plays a key role, therefore this study was focused on
investigating HSA adsorption behavior on differently modified PET
surfaces. PET is commonly used as a biomaterial for vascular grafts
and by modifying its surface properties with selected polysaccha-
rides (chitosan, fucoidan and chitosan sulfate) that possess specific
biological activities; improvement of biocompatible properties was

0927-7757/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfa.2010.03.003
 T. Indest et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 360 (2010) 210–219
expected. Hence protein HSA adsorption was monitored with a
quartz crystal microbalance with dissipation unit (QCM-D) on mod-
ified PET systems; PET-H/chitosan, PET-H/chitosan/fucoidan and
PET-H/chitosan/chitosan sulfate.
QCM is an efficient, high-resolution mass sensing technique
which can additionally provide information about energy dissi-
pating properties of the bound surface mass. It is one of a few
techniques that can be used to give a direct observation of the
adsorption process in situ. Thus, it is a suitable method for protein
adsorption studies at the solid/liquid interface [16,17].
2. Materials and methods
2.1. Materials
2.1.1. PET foil
The foil was Mylar® polyethylene terephthalate (PET) foil, of
thickness 175 ␮m.
2.1.2. PET film pre-treatment
PET foils were immersed in 98% ethanol and cleaned in ultra-
sonic bath for 10 min, washed thoroughly with demineralised
water and air dried.
2.1.3. Hydrolysis procedure
The PET foils were hydrolyzed in 4 M NaOH solution for 35 min
at 70 ◦ C. After that, the foils were immersed in 1 M HCl for 10 min
to neutralize the NaOH and to stop the hydrolysis. The foils were
air dried after rinsing with large amounts of demineralised water.
2.1.4. Spin coating
1 wt.% of PET foil was added to 1,1,2,2-tetrachloroethane (Fluka,
86960) and heated (T ≈ 150 ◦ C) until the foil was fully dissolved.
After cooling, the solution was filtered through a 0.2 ␮m Acrodisc
GHP filter. 30 ␮L of solution was spread on a 14 mm silica quartz
crystal and spin coated at maximum 2000 rpm for 60 s using a SCS
P-6708 spin coater.
2.1.5. QCM-D crystals
The quartz crystals (supplied by Q-Sense AB, Gothenburg,
Sweden) were AT-cut, equipped with gold plate electrodes and
sputtered with silica on the active surface. The fundamental fre-
quency of these quartz crystals was f0 ≈ 5 MHz and their sensitivity
constant C was 0.177 mg/m2 Hz.
2.1.6. Chemical compounds
All chemicals used were of analytical grade and used with-
out further purification. Solutions were prepared at least 24 h
before measurements, using Milli-Q water. The pH was adjusted
with NaOH (1 M, 2.5 M) and HCl (0.01 M, 0.1 M). Chitosan from
crab shells with low molecular weight (Aldrich, 448869), 75–85%
deacetylated, was used. Chitosan solutions were prepared as 0.2 g/L
in 1% (w/v) acetic acid with 140 mM NaCl. Fucoidan from Fucus
vesiculosus (Fluka, 47865) was used and prepared in concentra-
tions of 0.2 g/L in 140 mM NaCl aqueous solution. Chitosan sulfate
(15.8 ± 0.5% S) was prepared and synthesized from chitosan using
the procedure described in [18]. Chitosan sulfate solutions were
prepared at a concentration of 0.03 g/L in 140 mM NaCl aqueous
solution.
The protein human serum albumin (HSA) was a commercial
product, purchased from Sigma (ref. A1653), and used without fur-
ther purification. Solution of human serum albumin (0.05 g/L HSA)
was prepared in phosphate buffer saline (PBS; 6.44 mM KH2 PO4 ,
8 mM Na2 HPO4 , 140 mM NaCl). After dissolution the HSA the solu-
tion was left overnight at around 8 ◦ C (in refrigerator). Then the pH
211
was adjusted to 7.4 with NaOH. The solution was stored at 8 ◦ C and
used within a week.
2.1.7. Adsorption
The adsorption of chitosan and fucoidan onto PET-H films was
measured at pH 5, and of chitosan sulfate at pH 7.4, in all cases
from 140 mM NaCl solutions. The resulting adlayers of polysaccha-
rides were then rinsed with phosphate buffer saline (PBS; 6.44 mM
KH2 PO4 , 8 mM Na2 HPO4 , 140 mM NaCl; pH 7.4).
Before HSA adsorption, the initial baseline was established with
the adlayers on the quartz crystal immersed in PBS. Then a solution
of 0.05 g/L HSA in PBS (pH 7.4) was introduced into the QCM cell
and adsorption was allowed to proceed until equilibrium uptake
was reached. After this, the surface was rinsed with PBS solution in
order to test the stability of the adsorbed protein layer. The same
procedure was used also when measuring HSA adsorption on PET
surfaces without adlayers.
2.2. Analytical methods
2.2.1. Quartz crystal microbalance
The adsorption behavior of protein HSA on non-hydrolyzed PET
and hydrolyzed (PET-H) films as well as on differently modified PET
films (chitosan, fucoidan and chitosan sulfate) was studied with
a quartz crystal microbalance, the QCM-D E4 instrument from Q-
Sense AB, Gothenburg, Sweden. Adsorption measurements were
done under stagnant (no flow) conditions, with stepwise additions
of adsorbent every 20 min. The added volume amounted to 0.5 ml
at 0.5 ml/min flow rate. Measurements were performed at pH 7.4,
an ionic strength of 154 mM of salts (PBS) and a temperature of
23.4 ◦ C.
To reduce the influence of swelling on the measurement, the
spin-coated PET films were conditioned for about 20 h in PBS solu-
tion at pH 7.4 prior to protein HSA adsorption measurement. PET
films with one or two adsorbed layers (chitosan, chitosan/fucoidan
and chitosan/chitosan sulfate) were further used without drying
in between, only rinsing with PBS solution prior to protein HSA
adsorption.
The resonant frequency (f0 ≈ 5 MHz) of the crystal decreases
when additional mass is adsorbed on its surface. If the adsorbed
layer is rigid, evenly distributed and if its mass is much smaller
than the mass of the quartz crystal, then the decrease in frequency
(f) is proportional to the adsorbed mass. The adsorbed mass m
can be calculated from Sauerbrey equation:
C · f
m = − (1)
n
where C is a constant that describes the sensitivity of device to
changes in mass and n is the overtone number.
Frictional losses occur in the crystal and the adsorbed material
lead to a damping of the oscillation with a decay rate of the ampli-
tude that depends on the viscoelastic properties of the material.
The dissipation factor D is given by:
Ediss
D= (2)
2Estor
where Ediss is the total dissipated (lost) energy during one oscil-
lation cycle and Estor is the total energy stored in the oscillator.
The QCM-D instrument measures the change in dissipation factor
(D = D − Do ) when the material is adsorbed, where Do is the dis-
sipation factor of the pure quartz crystal immersed in the solution.
The samples used in QCM experiments are described in
Table 1.
212 T. Indest et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 360 (2010) 210–219
Table 1
Designation of spin-coated samples and samples with adsorbed components mon-
itored with QCM-D prior to HSA adsorption.
Sample Description
PET Non-hydrolyzed PET, spin coated on SiO2 crystal
PET-H Hydrolyzed PET, spin coated on SiO2 crystal
PET-HC PET-H sample with adsorbed chitosan
PET-HCF PET-H sample with adsorbed chitosan and fucoidan
PET-HCSH PET-H sample with adsorbed chitosan and chitosan sulfate
2.3. XPS analysis
XPS measurements were recorded with a Kratos Analytical AXIS
165 electron spectrometer using a monochromated Al K␣ X-ray
source. Acquisition was performed at 100 W, using a low-resolution
setting (1 eV step and 80 eV analyzer energy) for wide spectra, and a
high-resolution setting (0.1 eV step and 20 eV analyzer energy), for
detailed analysis. To distinguish the adsorbed components on spin-
coated PET films, surface elemental compositions were obtained
from wide spectra and high-resolution peaks of carbon C 1s, oxygen
O 1s, nitrogen N 1s and sulfur S 2p. The pressure in the analysis
chamber was lower than 1 × 10−6 Pa during the experiments. All
spectra were collected with an electron take off angle of 90◦ from
sample analysis areas less than 1 mm in diameter. Each sample was
measured on at least two locations.
2.4. AFM investigation
Atomic force microscopy (AFM) was performed with Nanoscope
IIIa multimode scanning probe microscope (Digital Instruments,
Santa Barbara, CA) and the images were scanned in tapping mode
using silicon cantilevers. No image processing except flattening was
made. The scanned image size was 5 ␮m × 5 ␮m.
3. Results and discussion
3.1. Characterization of spin-coated PET films (PET and PET-H)
3.1.1. Surface elemental composition
In previous publication [19] the results of XPS analysis per-
formed on spin-coated non-hydrolyzed (PET) and on hydrolyzed
(PET-H) films were published and discussed in detail. The results
are briefly summarized in Table 2, which shows the results from
XPS analysis of the high-resolution C1s peaks of carbon (C1, C2,
C3, C4) and oxygen in the non-hydrolyzed (PET) and hydrolyzed
(PET-H) films.
There is virtually no change in the percentage of C–O carbon
(C2) after hydrolysis, although one would expect this to increase
because hydrolysis of an ester bond in PET should result in the for-
mation of a terminal hydroxyl group (–CH2 CH2 OH). There is also
no change in the O–C O carbon (C4), indicating that the carboxyl
groups remain in the PET after hydrolysis. However, there is a sub-
stantial reduction in the O1 oxygen which occurs in ester groups:
reduction from 47 to 35 atom%, i.e., by 25%, and at the same time
the O2 oxygen increases from 53.3% to 62%, i.e., by 16%. This clearly
reflects that the number of free carboxyl groups in the PET has
increased after the treatment in alkali.
The rather small difference in chemical composition between
spin-coated PET and PET-H films indicated by XPS analysis could
Table 2
The results of high-resolution spectra fittings of carbon (C1, C2, C3, C4) and oxygen (O1, O2) for non-hydrolyzed (PET) and hydrolyzed (PET-H) films.
Sample C–C (C1) [at%] C–O (C2) [at%]
PET [19] 63.2 19.5
PET-H [19] 62.1 20.2
be explained by relatively hydrophobic (–CH2 CH2 OH) group that
turns inwards during film preparation or that the ester group at the
other end of the (–CH2 CH2 OH) is also hydrolyzed, so that ethylene
glycol that is formed is easily washed away. Nevertheless differ-
ences in frequency shift and dissipation between these samples
upon HSA adsorption were clearly seen in the QCM measurements.
These results are discussed below.
3.2. Characterization of PET films modified with chitosan,
fucoidan and chitosan sulfate
The results of XPS analysis performed on PET-H films with
adsorbed components (chitosan, fucoidan, chitosan sulfate) are
reported in detail in previous publications [18,19] and are sum-
marized in Table 3. Since nitrogen is the most important peak for
protein HSA determination, it is important to know how much
nitrogen was initially present on the differently modified PET-H
films. The nitrogen content was the highest for chitosan sulfate
(PET-HCSH) sample and amounted to 2.8%, followed by the chi-
tosan (PET-HC) sample with 1.4%. There was some nitrogen present
in the fucoidan (PET-HCF, 0.9%), however, it was difficult to distin-
guish contributions to the nitrogen content from different layers.
The fucoidan layer might have been too thin and consequently, the
underlaying chitosan layer was detected.
3.3. Adsorption of HSA on modified spin-coated PET films
3.3.1. Adsorption of HSA on PET and PET-H films
The adsorption of HSA on spin-coated non-hydrolyzed PET and
hydrolyzed PET-H model films, was studied. Fig. 1a and b shows
the frequency shift and dissipation changes as a function of time
for HSA adsorption on PET and PET-H films. The adsorption of HSA
was performed at repeated additions of HSA solution until equilib-
rium uptake was reached. Fig. 1a shows the frequency shift for HSA
adsorption on PET and PET-H spin-coated films. Adsorption equilib-
rium was reached at f = −27.1 Hz for PET/HSA and at f = −18.5 Hz
for PET-H/HSA, the difference in frequency shift corresponds to a
reduced adsorbed mass of about 31.7% on hydrolyzed (PET-H) in
comparison to PET.
The arrows in Fig. 1a show the starting point of rinsing with PBS
solution after protein adsorption, and it can be seen that frequency
remains stable, indicating that no desorption of HSA occurred from
either the PET or the PET-H layers.
More protein was adsorbed on the more hydrophobic PET sur-
face than on the hydrolyzed (PET-H) surface. These results are in
agreement with other studies of protein adsorption, which gen-
erally indicate that hydrophobic surfaces tend to adsorb larger
amounts of proteins than hydrophilic ones. Wannenberger et al.
[20,21] found a decrease of the adsorbed amount of the enzyme
lipase with increasing surface wettability of the substrate and
Andrade et al. [22] as well as Norde [23], reported hydrophobic
surfaces to adsorb more protein than hydrophilic ones. Fig. 1b
shows energy dissipation in PET/HSA and PET-H/HSA layers as a
function of time. The HSA adsorption induces moderate energy
dissipation. The changes in dissipation were smaller for the
PET/HSA sample (D = 0.92 × 10−6 ) than for the PET-H/HSA sam-
ple (D = 1.3 × 10−6 ). Dissipation was larger in the PET-H/HSA layer
than in the PET/HSA layer, indicating that the adsorbed HSA layer
on PET-H was slightly thicker and less compact than on PET. The
C O (C3) [at%] O–C O (C4) [at%] (O C–O) O1 [at%] (O C–O) O2 [at%]
– 17.4 46.7 53.3
– 17.7 34.8 65.2
 T. Indest et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 360 (2010) 210–219 213

Table 3
The results of surface elemental concentrations obtained from XPS wide spectra of modified PET-H films with chitosan (PET-HC (0.2)), chitosan/fucoidan (PET-HCF(0.2)) and
chitosan/chitosan sulfate (PET-HCSH(0.03)).

Sample O 1s [at%] C 1s [at%] N 1s [at%] S 2p [at%] Si 2p [at%] Na 1s [at%] Cl 2p [at%]

PET-HC [19] 25.1 72.0 1.4 0.2 1.1 0.1 0.1
PET-HCF [16] 26.3 70.1 0.9 0.3 1.1 0.9 0.4
PET-HCSH [16] 29.8 64.3 2.8 1.4 1.5 – 0.2

explanation for this might be found in the conformation of HSA
molecules in the adlayer or in binding/trapping of more water
[14,17,24] in the HSA film adsorbed on hydrolyzed PET (PET-H).
However, it is more likely that both events contributed to the dis-
sipation changes.
The larger frequency shift and the smaller shift in dissipation
for PET/HSA sample indicate that more adsorbed mass was more
rigidly distributed and denser packed on the surface than on the
PET-H/HSA sample.
The D/f curves in Fig. 2 compare the behavior of HSA layer
on PET and PET-H surfaces. The D/f plot gives an indication
of how the new added mass affects the adsorbed layer struc-
ture. The spacing of data points becomes more distant when the
adsorption kinetics is faster [17]. For both samples there was ini-
tial slow adsorption of HSA, as indicated by the high density of
data points, then the distance between data points increased, indi-
cating faster adsorption up to about f = −6 Hz for adsorption of
HSA on PET-H sample and slightly above f = −12 Hz for adsorp-
tion on PET/HSA. Then there was again a slower process of diffusion
during which f and D slowly increased. The slope of the curve
for PET-H/HSA is slightly steeper and hence the protein layer was
more loosely bound to the surface than on the PET/HSA surface

Fig. 1. Change in frequency (third overtone) (a) and dissipation changes (third overtone) (b) as a function of time for HSA (0.05 g/L HSA, pH 7.4) adsorption on non-hydrolyzed
(PET) and hydrolyzed PET (PET-H) films. The points where the rinsing with phosphate buffer (154 mM salts, pH 7.4) started are marked with “PBS”.
214 T. Indest et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 360 (2010) 210–219
Fig. 2. Change in the dissipation factor as a function of change in frequency (third overtone) occurring at multiple adsorption steps of HSA (0.05 g/L, pH 7.4) on PET(grey line)
and PET-H (black line) film.
where the protein layer thus was more rigid and more densely
packed.
3.3.2. Adsorption of HSA on modified spin-coated PET films
The adsorption mechanism and the stability of adsorbed lay-
ers of chitosan, fucoidan and chitosan sulfate on PET films was
investigated in detail earlier [18,19]. The general conclusions from
these studies of importance for the adsorption of HSA are that even
at high ionic strength (up to 140 mM NaCl) electrostatic interac-
tions are not totally screened, but high ionic strength and high
concentrations of polymer result in denser and thicker adlayers.
Chitosan/fucoidan films are thinner and more compressed than chi-
tosan/chitosan sulfate layers, in which large amounts of adsorbed
chitosan sulfate form a loose and thick adsorbed layer.
Fig. 3 shows the changes in frequency (Fig. 3a) and changes in
dissipation (Fig. 3b) as a function of time for protein HSA adsorp-
tion on non-hydrolyzed (PET), hydrolyzed PET film (PET-H) and
films modified by adsorption of chitosan (PET-HC), fucoidan (PET-
HCF) and chitosan sulfate (PET-HCSH). The values for the changes
in frequency and dissipation at adsorption equilibrium for HSA
adsorption on PET, PET-H and on modified PET-H films (PET-HC,
PET-HCF, PET-HCSH) are summarized in Table 4.
Clear differences can be observed between HSA adsorption
behavior on hydrolyzed and non-hydrolyzed PET substrates, as
well as between adlayers of chitosan, chitosan/fucoidan and chi-
tosan/chitosan sulfate.
The frequency shift for HSA at adsorption equilibrium on the
PET-HC layer amounts to f = −17.4 Hz, which is only slightly lower
than the value f = −18.5 Hz for PET-H/HSA. This rather minor dif-
Table 4
The average values of frequency shift (third overtone, f3 [Hz]) and energy dis-
sipation changes (third overtone, D3 ) at adsorption equilibrium are presented
for protein adsorption on non-hydrolzed (PET), hydrolyzed film (PET-H) and on
modified PET films by chitosan (PET-HC), chitosan/fucoidan (PET-HCF) and chi-
tosan/chitosan sulfate (PET-HCSH):.
Sample f3 (Hz) max D3 × 10−6
PET/HSA −27.1 0.92
PET-H/HSA −18.5 1.3
PET-HC(0.2)/HSA −17.4 0.7
PET-HCF (0.2)/HSA −7.1 0.3
PET-HCSH (0.03)/HSA −1.3 −0.7
ference can be explained by referring to the previously published
[19] AFM analysis of chitosan-covered hydrolyzed PET (PET-HC),
which shows that chitosan does not cover the whole PET-H sur-
face, but rather forms some kind of blobs on its surface (see Fig. 4c).
Thus, HSA was adsorbed on chitosan only partly covering PET-H
surface as well as on uncovered parts of PET-H film. This arguably
explains why such a small difference in frequency change after HSA
adsorption between the PET-H and PET-HC surfaces was observed.
In case of fucoidan (PET-HCF) the frequency shifts after HSA
adsorption amounted to f = −7.1 Hz at adsorption equilibrium
(∼65 min). Thus, only a small amount of HSA was adsorbed on this
surface. Previously published AFM results [19] show that fucoidan
covers the PET-HC surface homogenously. Hence the protein was
adsorbed on a fucoidan layer covering the PET surface.
In the case of chitosan sulfate (PET-HCSH) there was almost neg-
ligible change in the frequency by HSA adsorption (∼30 min). This
indicates that protein did not adsorb to the sample’s surface. It was
found previously by Indest et al. [18], in an investigation of chi-
tosan sulfate adsorption on PET-H, that a large amount of chitosan
sulfate is adsorbed, forming a thick and loose adlayer fully cover-
ing the sample’s surface, thus leaving no sites available for protein
HSA to adsorb. The results show that both fucoidan and chitosan
sulfate when adsorbed on chitosan reduce the amount of protein
adsorbed, but there are differences in their efficiency.
To produce protein-resistant biomaterial surfaces, it is clear
that one must design systems where repulsive interactions are
maximized and attractive ones are minimized [25]. The purpose
of using chitosan as a sublayer was to improve the adsorption
of the two anionic polysaccharides to the PET-H surface. Both
fucoidan and chitosan sulfate carry negative (sulfate) charges that
can be expected to contribute to repulsion of the approaching neg-
atively charged HSA molecules, at least at low or moderate ionic
strength. Another well-known repulsive mechanism is steric repul-
sion, which requires that a solvent-swollen adsorbed polymer layer
interacts more strongly with the solvent than with the adsorbate
molecules, and also that the adsorbed layer is thick enough so that
van der Waals interactions between the underlying surface and
the adsorbate are small [25,26]. QCM-D studies of chitosan sulfate
adsorbed on chitosan [18] show that under the conditions used in
this investigation, chitosan sulfate forms a water-swollen layer that
is expected to well fulfill these conditions, while the fucoidan layer
is thinner. Nevertheless, although as noted above, electrostatic
 T. Indest et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 360 (2010) 210–219 215

Fig. 3. Change in frequency (f3 , third overtone) (a) and change in dissipation (D3 ) (b) as a function of time for HSA adsorption (0.05 g/L HSA, PBS solution) on non-
hydrolyzed (PET)—grey line, hydrolyzed PET film (PET-H)—black line, and modified films with chitosan (PET-HC, 0.2 g/L)—violet line, fucoidan (PET-HCF, 0.03 g/L)—blue line;
and chitosan sulfate (PET-HCSH, 0.03 g/L)—green line (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.).

interactions may play some part even at the high ionic strength
used, it is obvious from comparison of the three surfaces that steric
repulsion is a predominant factor preventing the adsorption of
HSA.
Washing with PBS solution was performed after each HSA
adsorption experiment and did not result in frequency increase,
indicating that no desorption of HSA occurred from any of the
modified PET samples.
Fig. 3b shows the dissipation changes (D) as a function of time
for HSA adsorption on PET, PET-H and films modified with chitosan
(PET-HC), fucoidan (PET-HCF) and chitosan sulfate (PET-HCSH). The
dissipation change at equilibrium was slightly larger for the PET-
H/HSA layer than for PET/HSA, and was lower than both for the
chitosan (PET-HC/HSA) layer (Table 4). In case of fucoidan (PET-
HCF/HSA) sample the dissipation change after protein adsorption
was very low and for the chitosan sulfate sample (PET-HCSH/HSA)
the dissipation decreased to a −0.7 × 10−6 . The negative dissipa-
tion value might indicate that, although small amount of HSA is
adsorbed on chitosan sulfate sample, HSA forms a compact layer
with sulfated chitosan from which water is expelled.
Dissipation indicates that rigidly attached adsorbed layers of
HSA were formed on PET and PET-HC samples but less so on PET-
H sample. The rather small dissipation changes on PET-HCF and
PET-HCSH samples correspond to minor frequency shifts, and con-
sequently almost no adsorbed mass of HSA on these samples. As
a rule, dissipation changes less than 1 × 10−6 indicate that the
adsorbed layer is sufficiently rigid so that the adsorbed mass can
be calculated from Sauerbrey equation (Eq. (1)) [27,28].
Since the results in Table 4 indicate that these conditions are
fulfilled for the HSA layers except for PET-H/HSA, the adsorbed
mass of HSA on the other layers was calculated using Sauerbrey
equation, yielding the frequency shift for PET sample corresponds
to 1.6 mg/m2 HSA on PET, 1.03 mg/m2 on PET-HC, 0.42 mg/m2 on
PET-HCF and 0.04 mg/m2 on PET-HCSH.
216 T. Indest et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 360 (2010) 210–219

Fig. 4. Height (left) and Phase (right) contrast AFM images of (a) spin-coated PET-H sample [19], (b) protein HSA adsorbed on PET-H, (c) PET-HC (0.2 g/L) sample [19] (d)
protein HSA adsorbed on PET-HC, (e) PET-HCF (0.2) sample [18], (f) protein HSA adsorbed on PET-HCF (g) PET-HCSH (0.03) sample [18], and (h) protein HSA adsorbed on
PET-HCSH sample.
T. Indest et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 360 (2010) 210–219 217

Fig. 4. (Continued ).
218 T. Indest et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 360 (2010) 210–219
Table 5
The average results of elemental surface concentration from XPS survey spectra
of hydrolyzed PET film (PET-H) and protein HSA adsorbed onto PET-H film (PET-
H/HSA).
Sample O 1s [%] C 1s [%] N 1s [%] Na 1s [%] Si 2p [%]
PET-H 27.0 72.6 – – 0.2
PET-H/HSA 23.3 71.4 2.5 0.2 2.2
3.4. Characterization of the adsorbed HSA on modified PET films
3.4.1. Surface chemical composition
The elemental composition of PET-H and PET-H film with
adsorbed HSA (PET-H/HSA) was determined by XPS analysis. The
results from XPS survey spectra of both samples are shown in
Table 5. There are clear differences in atomic composition between
pure PET-H film and film with adsorbed HSA. In XPS analysis nitro-
gen is the most important peak for determination of proteins, such
as HSA. The absence of nitrogen in the PET-H film and a signifi-
cant amount of nitrogen (2.5%) in the PET-H/HSA film confirm that
protein was adsorbed on the PET-H film surface. As expected, the
XPS results (survey spectra) of PET films modified with chitosan,
fucoidan and chitosan sulfate; summarized in Table 3, indicate high
nitrogen content. XPS analysis of these films after adsorption of HSA
was not performed, due to the difficulty to distinguish contribu-
tions from different adsorbed layers to nitrogen content, especially
when such small amounts of protein HSA were adsorbed on PET-
HCF and PET-HCSH samples.
3.5. Morphology
The topography of hydrolyzed PET-H film and of modified films
(chitosan, fucoidan, and chitosan sulfate) after protein HSA adsorp-
tion was studied by atomic force microscopy (AFM).
Fig. 4a shows height and phase contrast AFM images of the
hydrolyzed PET (PET-H) film. Its surface is smooth and uniform.
Fig. 4b shows that HSA was adsorbed onto the PET-H film in a grain
shape, completely covering its surface. The chitosan-coated (PET-
HC) sample shown in Fig. 4c exhibits uneven coverage of the PET-H
surface as chitosan formed some kind of blobs. Fig. 4d shows that
HSA when adsorbed on chitosan sample completely covered the
surface in a similar grainy form as on PET-H. On the films cov-
ered with fucoidan (PET-HCF, Fig. 4e) and with chitosan sulfate
(PET-HCSH, Fig. 4g), large white spots were observed on the height
image, indicating that the salt crystals (either NaCl or PBS) were
adsorbed on the surface. In AFM images of PET-HCF/HSA (Fig. 4f)
and PET-HCSH/HSA (Fig. 4h) samples it is very hard to distinguish
contributions from adsorbed protein layer, in agreement with the
very low amounts of adsorbed HSA indicated by the QCM-D mea-
surements.
4. Conclusion
The purpose of this study was to investigate how differ-
ent modifications of PET surface with selected polysaccharides
(chitosan, fucoidan, chitosan sulfate) influence on protein HSA
adsorption. Small differences in chemical composition between PET
and hydrolyzed PET (PET-H) films resulted in different adsorption
behavior of HSA. The adsorbed mass is more rigidly deposited and
denser packed on a PET/HSA surface than on a PET-H/HSA surface.
The adsorbed mass of HSA was about 31.7% lower on hydrolyzed
PET-H than on PET. This correlates well with other literature data
that indicate lower adsorption of HSA on a more hydrophilic sur-
faces; this was in this case achieved by alkaline hydrolysis of PET
surface.
PET film surfaces with adsorbed chitosan, fucoidan and chitosan
sulfate components adsorb less HSA, especially in the case of films
with fucoidan and chitosan sulfate. The coverage of the surface
has proven to be an important factor for protein adsorption and
when the surface is fully covered especially by chitosan sulfate,
protein adsorption does not occur. Thus it can be concluded that
Cl 2p [%]
surfaces, modified with negatively charged sulfated polysaccha-
0.2
0.4
rides (fucoidan, chitosan sulfate), are effective in preventing HSA
adsorption due to steric repulsion between polysaccharides and
HSA. The obtained reduced HSA adsorption is in agreement with
biocompatibility definition by which a protein adsorption should
be reduced in order to improve biocompatibility. Therefore the
decrease in non-specific protein adsorption can be the measure for
improved biocompatibility.
Acknowledgements
We gratefully acknowledge the support of Dr. Monika Österberg
regarding interpretation of AFM images, Ritva Kivelä and Marja
Kärkkäinen are acknowledged for performing AFM measurements.
Special thank goes to Dr. Leena-Sisko Johansson and to Dr. Joseph
Campbell for performing the XPS measurements and to Petri Myl-
lytie for the support regarding spin coating.
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